WO2003010507A2

WO2003010507A2 - System for intracellular process monitoring and in vivo drug screening

Info

Publication number: WO2003010507A2
Application number: PCT/IB2002/004257
Authority: WO
Inventors: Inhwan Hwang; Dae Heon Kim; Yong Jik Lee; Jing Bo Jin
Original assignee: Ahram Biosystems Inc.
Priority date: 2001-07-16
Filing date: 2002-07-16
Publication date: 2003-02-06
Also published as: AU2002339228A1; WO2003008536A2; KR100919637B1; KR20040015804A; WO2003010507A3; KR20030007221A

Abstract

This invention relates to a method for detecting characteristics related to intracellular trafficking and subcellular localization of a selected reporter protein. The invention has a wide spectrum of important applications including use in screens to detect compounds that modulate protein trafficking in vivo .

Description

SYSTEM FOR INTRACELLULAR PROCESS MONITORING AND IN VIVO DRUG SCREENING

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to Korean patent application No.10-

2001-0042886 as filed on July 16, 2001, the disclosure of which is incorporated herein by reference. The present application also claims benefit to U.S Provisional Application

Serial No. by Hwang, I. et al. as filed on July 16, 2002 with the above- captioned title, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION The present invention generally relates to methods for detecting intracellular movement (trafficking) of a selected protein inside cells. The invention further relates to methods for detecting compounds (screens) that modulate at least one of expression and trafficking of the protein in vivo. The invention has a wide spectrum of applications including use in the detection of drugs that impact protein trafficking to a pre-selected subcellular organelle.

BACKGROUND

The recent rapid development in biotechnology makes it possible to collect primary information on the whole genomic sequence and the expression pattern of the total cellular proteins of an organism. Genomics and Proteomics researches become new leading trends to discover valuable genes and proteins from the vast amount of the primary information and also to elucidate and use the correlations among such bio- information. In order to extract and use valuable information from such vast primary information, it is necessary to develop technologies that can selectively and quickly sort and detect the detailed processes of the complex biological phenomena, as realized in the present invention. Developing such technologies is one of the leading trends as found in various recent arts pertaining to the present invention.

The most important usage of the large amount of the useful information obtained from Genomics and Proteomics is for developing new drugs. Establishment of efficient and selective drug screening systems based on such useful bio-information is expected to play a major role in evolution of the biotechnology. Therefore, researches for developing new drug screening systems have been actively carried out recently.

Development of a new drug screening system depends basically on (1) discovery and establishment of a specific target related to a disease or a biological regulation process, and (2) development and optimization of an assay method to determine the effects of drug candidates on a specific target. In order to develop efficient drug screening systems, therefore, selective detection techniques must be developed first. Such detection techniques can be used to identify detailed causes of biological phenomena from the vast amount of the genetic and proteomic information, or to detect specific intracellular or intercellular processes related to such causes. These selective detection techniques provide fundamental basis not only for identifying specific targets for drug screening, i.e., biological factors causing specific phenomena, but also for establishing systematic assay methods that allow more selective drug screening.

The assay method to evaluate the effects of drug candidates on a specific target can be categorized into three classes. The first is a protein- or a molecule-based method in which the effect of the drug candidate on the activity of an enzyme or a receptor critical in the induction mechanism of a disease is directly observed to screen the drug candidates. The second is an organism-based method in which the effect of the drug candidate on the morphological or biochemical characteristics of an organism is observed to screen the drug candidates. The last is a cell-based method in which the effect of the drug candidate on the morphology of the cell or subcellular organelles, and expression, trafficking, and metabolism of proteins in the cell is observed to screen the drug candidates. The protein-based method has the advantage that the assay can be performed for a specific target protein. However, because the assay is conducted in vitro, it is not possible to examine various complex cellular factors. Therefore, it requires many additional time-consuming experiments before examining the drug candidate to a living organism. On the other hand, the organism- or cell-based method can detect the composite changes of the organism or the cell induced by the drug candidates. But it also requires additional time-consuming experiments to confirm that the drug candidate specifically affects the target gene or protein.

To overcome such problems of the prior drug screening methods, new technologies are recently under development using genetic transformation techniques. Genetic functions of a specific gene or biological functions of a protein expressed from the specific gene can be elucidated by examining the phenotype of the transformed organism or cell. Drug candidates can be more selectively screened by examining their effects on the specific phenotype of the transformed organism or cell. Previously, the radioisotope labeling method has been frequently used to selectively observe a specific phenomenon in a cell or an organism. Recently, fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein (REF) (Moris et al., 1974), and their derivatives (Evans, W098/21355) that can be expressed in the cell have been developed, and various researches have been reported using expression of these fluorescent proteins. Examples of using the fluorescent proteins include observation of the gene expression and subcellular protein localization (Chalfie and Prasher, US5491084), visualization of the subcellular organelles (Kost et al., 1998), visualization of the protein trafficking in the secretory pathway (Kaether and Gerdes, 1995), and visualization of the protein expression pattern in a plant cell (Hu and Cheng, 1995) and in Drosophila embryo (Davis and Viestra, 1998). The fluorescent proteins have been also used to develop new drug screening methods. Examples include a method using mutated organisms transformed to express a modified GFP (Ward and Chalfie, W095/21191), a method using a cell transformed to express a chimeric protein comprising a fluorescent protein and a transcription factor related to the activity of a cell surface receptor which regulates signal transduction (Harpold et al., US5401629), and a method using a cell transformed to express a chimeric protein comprising a fluorescent protein and the active site of the protein kinase (Thastrup et al., W096/23898).

Among these studies that utilize the expression of the fluorescent proteins, several examples showed that intracellular processes related to specific proteins can be selectively observed among the complex cellular processes by visualizing the expression and trafficking of a selected protein (Ward and Chalfie, W095/21191; Kost et al, 1998; Gilooly et al., 2000; Pih et al., 2000). Various researches have reported signal proteins that have trafficking signals targeting to specific subcellular organelles. For example, the chlorophyll-binding proteins are transported to chloroplast, the nuclear localization signal domain (NLS domain) of SV40 is targeted to the nucleus (Goldfarb et al., 1986), the peroxisome targeting motif SKL is targeted to peroxisome (Davis and Viestra, 1998), and FI- H⁺-ATPase is transported to mitochondria (Niwa et al., 1999). As in these examples, most of proteins in the cell are transported to specific subcellular organelles related to their functions. Therefore, it is possible to visualize and observe intracellular trafficking of a specific signal protein (Harpold et al., US5401629; Kost et al., 1998). Phospholipid-specific intracellular processes can also be observed by expressing phospholipid-binding proteins linked to a fluorescent protein. For example, PLC-Delta PH (Stauffer et al., 1998), AtPH, and FAPP1 (Dowler et al., 2000) domains are known to bind specifically to PI(3,4)P2, PI(3)P, and PI(4)P, respectively. A chimeric protein having the PH domain and GFP was shown to be translocated to the plasma membrane when expressed in a cell (Kost et al., 1998).

It would be useful to have methods for detecting compounds that modulate intracellular movement of proteins (trafficking) . Particular methods would be able to monitor changes in protein localization in vivo. It would be especially useful to have methods that express the protein and provide for reliable in vivo imaging of the protein inside living cells.

SUMMARY OF THE INVENTION

The invention generally relates to methods for detecting intracellular movement (trafficking) of at least one selected protein inside cells. Additional invention methods involve screening compounds for capacity to modulate at least one of expression and trafficking of the protein in vivo. The invention has many important uses including detecting compounds that modulate protein trafficking to at least one preselected subcellular organelle. The invention provides useful methods for detecting trafficking of at least one selected (reporter) protein inside living cells. Particular reporters of interest include at least one "signal" sequence and at least one detectable tag. Preferred signal sequences help shuttle the protein to at least one other intracellular location. More particular reporters include at least one detectable tag, usually a fluorescent (or potentially fluorescent) sequence, that is operably and covalently attached to the signal sequence in- frame, thereby labeling the reporter protein. Particular signal sequences of interest include or consist of amino acid residues that are known to help shuttle the reporter protein to at least one subcellular organelle. As discussed in more detail, such tagged reporter proteins can be used to screen compounds for capacity to modulate (increase or decrease) trafficking to one or more of the organelles.

Practice of the invention generally involves detecting trafficking of at least one of the selected reporter proteins inside cells. For instance, and in one embodiment, the signal sequence of a reporter include or consist of recognition site(s) that facilitate movement of the reporter protein to a sub-cellular organelle. An example of such proteins are characterized herein as Group I signal proteins (see Figure 1). In another embodiment, the proteins include or consist of amino acid sequences that are recognized by and typically contacted by endoplasmic reticulum (ER). Preferred examples of such proteins are featured herein as Group II signal proteins (see Figure 4). These reporter protein groups, as well as others discussed below, can be used as important tools to detect compounds that modulate such trafficking in vivo. Such proteins can also be used to confirm trafficking activity in cases in which such function is known or suspected.

The present invention provides important advantages.

For instance, the invention provides, for the first time, recognition that operably linked fusions of a signal protein and a detectable tag can be used to screen compounds for capacity to modulate intracellular trafficking in vivo. That is, the invention is one of general application in which a wide spectrum of suitable fusion proteins can be made and used to detect compounds for capacity to increase, decrease or preserve existing intracellular protein trafficking to one or more pre-selected subcellular organelles. Thus in one embodiment, any of the Group I or II protems can be fused to at least one detectable tag to create useful reporter protein fusions. Such protems can be used, for instance, to detect compounds for ability to modulate trafficking to at least one of the nucleus, chloroplast, mitochondria, and endoplasmic reticulum. Most reporter proteins in accord with the invention will include signaling features that provide for trafficking of the reporter to a single pre-selected subcellular organelle. Also envisioned however are reporters with multiple signal proteins (eg., two or three) that can provide trafficking to more than one of pre-selected organelle.

Additional benefits are provided by the invention.

For instance, and in one embodiment, each reporter protein includes at least one fluorescent sequence preferably bound in-frame, and either directly or indirectly, to at least one signal protein. In such embodiments, the fluorescent sequence is intended to provide a highly sensitive and reproducible tag that can be used to detect minute amounts of trafficked protein inside cells. Such advantages enhance the sensitivity, selectivity and reproducibility of the invention particularly in screens that are designed to detect compounds with potential trafficking activity. Additionally, the invention is fully compatible with existing screening platforms including what is often referred to as "high" or "ultra-high" throughput strategies. For instance, the invention can provide an important and valuable in vivo confirmation of such strategies where trafficking activity of a subject compound is known or suspected.

Accordingly, and in one aspect, it is an objective of the present invention to provide a method for detecting trafficking and localization of a selected protein inside a cell. In one embodiment, such a method includes introducing and expressing at least one type of, preferably less than about five, more preferably one type of reporter protein inside cells and detecting the cellular distribution of that reporter to detect trafficking and localization of the desired protein within the cells. The general detection method can be used as a screening platform to detect and optionally quantify trafficking activity of one or more compounds to be tested. Thus in one invention embodiment, the cell expressing the reporter protein can be contacted with at least one desired compound to be tested, preferably less than about fifty of such compounds, more preferably less than about ten to fifteen of such compounds, even more preferably one compound to be tested. The compound to be tested is added to the cells under conditions conducive to providing a protein trafficking effect. That is, the compound can be added to cultured cells before, during or after introduction of the reporter protein. In this example of the invention, cellular distribution of the reporter protein is visualized in the presence of the compound to detect any modulation of protein trafficking inside the cells. Typical practice of the invention involves comparison with a suitable control eg., as when water or buffer is added to the cells in lieu of the tested compound.

The invention further encompasses diverse and complex intracellular processes and a detailed method for selectively screening compounds (eg., chemicals) that affect a specific intracellular process based on the above detection method. To accomplish this obj ect, the present invention provides :

(1) an efficient method for preparing a transformed cell to express a reporter protein comprising a signal protein having a trafficking signal targeting to a specific intracellular organelle and a fluorescent protem linked thereto,

(2) detailed methods for preparing various recombinant genes, each comprising a gene encoding a signal protein and a gene encoding a fluorescent protein linked thereto, and

(3) methods for constructing recombinant plasmids that can be used to efficiently express the reporter proteins in a cell.

The present invention also provides a systematic method for transforming a cell with the recombinant plasmid and expressing efficiently the reporter protein in the transformed cell. Furthermore, the present invention provides a detailed method for monitoring details of the intracellular distribution of the reporter protein in a living cell by observing the fluorescence image either continuously or step by step during the expression and trafficking processes. In addition to providing the efficient systematic method for measuring the spatial distribution specific to the expression and trafficking processes of a selected protem in the transformed cell, it is another objective of the present invention to provide a method for selectively screening drug candidates that affect a specific intracellular process in the transformed cell. In order to expand and systemize the usages of the selective detection method for the intracellular processes and the drug screening method based on this detection method, the present invention provides recombinant genes for diverse signal proteins that direct targeting to specific intracellular organelles such as nucleus, mitochondria, chloroplast, peroxisome, plasma membrane, endoplasmic reticulum, Golgi apparatus, storage vacuole, lytic vacuole, prevacuolar compartment, etc. In addition, the present invention aims to provide an optimized overall procedure from the construction of the recombinant plasmids to the preparation of the transformed cells.

Practice of the invention provides additional benefits.

For instance, it establishes a method for selectively detecting specific characteristics related to intracellular trafficking and localization of a selected protein and a method for selectively screening chemicals that affect such specific cellular characteristics of the selected protein.

Also, it provides detailed methods for preparing the transformed cells that can express diverse reporter proteins targeting to various subcellular organelles. Therefore, a fundamental basis is provided for using more systematically and more thoroughly the selective detection method for the intracellular processes and the selective drug screening method based on this detection method.

The invention also establishes a method for determining the detailed effects of chemicals by examining the effects of several known chemicals on the intracellular processes. In further detail, the present invention provides (1) the method for selectively screening chemicals that affect the intracellular trafficking and localization of a selected protem, (2) the method for screening chemicals that inhibit or enhance the transcription or translation process by monitoring the effects of the chemicals on the expression of the reporter protein, and (3) the method for screening cytotoxic chemicals by observing the effect of chemicals that cause deformation, damage, or disruption of subcellular cellular organelles.

Other uses and advantages of the invention are disclosed below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows schematic diagrams of the reporter protems targeting to the subcellular organelles enclosed by membrane, which are classified as Group I proteins in the present invention.

Figure 2 shows fluorescence images showing the expression of the Group I reporter proteins in the cell, wherein

(a) shows that AtOEP7:GFP is localized to the envelope of chloroplast, wherein the red fluorescent signal is the auto-fluorescence signal of chloroplast and the yellow fluorescence signal is an merged image of the auto-fluorescence signal of chloroplast and the green fluorescence signal of the reporter protein;

(b) is a photograph where the red auto-fluorescence of chloroplast in (a) is eliminated by using a filter;

(c) shows that Rubisco small subunit (RbcS) is localized in the stroma of chloroplast;

(d) shows that Cab:GFP emits in the chloroplast;

(e) shows that Rubisco Activase (RA) linked to GFP is localized in the chloroplast;

(f) shows that FI- H^-ATPase linked to GFP is localized in the mitochondria; (g) shows that peroxisome targeting signal (SKL) linked to GFP is localized in the peroxisome envelope; and

(h) shows that nuclear localization signal (NLS) linked to GFP is localized in the nucleus.

Figure 3 shows a photograph of a Western blot analysis of the expressed AtOEP7:GFP, wherein T, S, and M indicate the total protein extract, the soluble fraction, and the membrane fraction, respectively.

Figure 4 shows schematic diagrams of the reporter protems targeting to the subcellular organelles by endosomal trafficking, which are classified as Group II protems in the present invention. Figure 5 shows fluorescence images showing the expression of the Group II reporter proteins in the cell, wherein

(a) shows that H⁺-ATPase:GFP is localized in the plasma membrane;

(b) shows the fluorescence image of sialtransferase (ST) translocated in the Golgi apparatus, wherein the red fluorescence signal is the auto-fluorescence signal of chloroplast;

(c) shows that the reporter protem BiP:RFP is localized in the lumen of the endoplasmic reticulum;

(d) shows that 500:GFP:KKXX is localized in the membrane of the endoplasmic reticulum; (e) shows that 526:GFP is localized in the storage vacuole;

(f) shows that Chi-n:RFP:Chi-c is localized in the storage vacuole;

(g) shows that Chi-n:GFP, in which the carboxyl terminus of the signal protein, chitinase, is not present, fails to translocate in the storage vacuole but appears as speckles in the endoplasmic reticulum; and (h) shows that SPO:GFP is distributed evenly in the lytic vacuole.

Figure 6 shows schematic diagrams of the reporter proteins binding specifically to phospholipids, which are classified as the Group III proteins in the present invention.

Figure 7 shows fluorescent images showing the expression of the Group III reporter proteins, wherein

(a) shows that the reporter protem GFP:EBD emits fluorescence at the outer membrane of vacuole, indicating that phosphatidylmositol 3 -phosphate (PI(3)P) is present on the outer membrane of vacuole;

(b) shows that GFP:FAPP1 emits fluorescence in the plasma membrane indicating that phosphatidylmositol 4-phosphate (PI(4)P) is present in the plasma membrane; and (c) shows that GFP:PH emits fluorescence in the plasma membrane, indicating that phosphatidylmositol 4,5-diphosphate (PI(4,5)P2) is present in the plasma membrane.

Figure 8 shows photographs representing the inhibitory effect of wortmannin on the intracellular trafficking of a Group I reporter protein RbcS:GFP. Wortmannin is known to be an inhibitor of PI(3)P and PI(4)P.

(a) shows that RbcS:GFP is targeted correctly to the chloroplast in the control protoplast, as expected.

(b) and (c) show that in the presence of wortmannin, the green fluorescence signal is not located in the chloroplast but appears as speckles or aggregates. Figure 9 shows photographs representing the effect of a chemical on the intracellular trafficking of a Group II reporter protein, wherein

(a) and (b) show that the green fluorescence signal of 500:GFP:KKXX is observed as numerous networks in the control protoplast; and

(c) and (d) show that in the presence of BafAl, the green fluorescent signal is observed as ring patterns in the plasma and vacuolar membranes.

Figure 10 shows photographs showing the change induced by a specific inhibitor upon co-expression of two reporter proteins, wherein

(a) and (b) show the green and red fluorescent signals in the control protoplast, respectively; (d) shows that treatment with brefeldin A does not affect BiP;

(e) shows that treatment with brefeldin A disrupts the Golgi apparatus and thus ST is transported to the endoplasmic reticulum instead of the Golgi apparatus; and

(c) and (f) show that the control protoplast and the brefeldin A-treated protoplast represent distinctive difference in their fluorescence images. Figure 11 shows photographs that visualize the effects of brefeldin A disrupting the subcellular organelles, wherein (a) shows that BiP:RFP is distributed along the structure of the endoplasmic reticulum in the control protoplast;

(c) visualizes that the disrupted structure of the endoplasmic reticulum in the presence of brefeldin A; and (b) and (d) show the protoplasts in (a) and (c) observed under bright field.

Figure 12 shows photographs representing the inhibitory effect of chemicals on Group III reporter proteins, wherein

(a) shows that the green fluorescence signal in the control protoplast is located on the vacuolar membrane along the distribution of PI(3)P which binds to EBD, but in the presence of wortmannin or LY294002, the green fluorescence signal is distributed throughout the cytosol; and

(b) shows that the distribution of the fluorescence signal is not affected by the above chemicals when the same experiments were performed with GFP:EBDC1358S, in which the amino acid residue 1358 of EBD was mutated. Figure 13 shows photographs showing the variation in the intensity of the fluorescent signal induced by an expression inhibitor, wherein

(a) shows that the intensity of the green fluorescence is decreased in the cycloheximide-treated protoplast compared to that in the control protoplast of (b); and

(c) and (d) show the auto-fluorescence signal of the chloroplast presented for relative comparison of the cell growth and the metabolism conditions.

DETAILED DESCRIPTION OF THE INVENTION

As discussed, the invention relates to a method detecting intracellular movement (trafficking) of a selected reporter protein inside cells. In one example, the invention provides methods for screening compounds for capacity to modulate at least one of expression and trafficking of the protein in vivo. Particular use of the invention involves screening compounds for capacity to modulate protein trafficking to a subcellular organelle.

As also discussed, the present invention provides a method for detecting specific characteristics related to trafficking and localization of a selected protein in a cell. In one embodiment, the method comprises at least one of:

(a) preparing a recombinant gene comprising a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protein linked thereto; (b) preparing a recombinant plasmid comprising the recombinant gene with a promoter and a terminator operably linked thereto so that the recombinant gene can be expressed in the cell;

(c) transforming the cell with at least one recombinant plasmid prepared in step

( );

(d) expressing the reporter protein comprising the signal protein and the fluorescent protein in the transformed cell; and

(e) detecting the cellular distribution of the reporter protein by monitoring the fluorescence image of the transformed cell during the expression or trafficking, or thereafter.

In a second aspect, the present invention provides a method for screening chemicals that affect specific characteristics related to trafficking and localization of a selected protein in a cell. In one embodiment, the method comprises at least one of:

(a) preparing a recombinant gene comprising a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protein linked thereto; (b) preparing a recombinant plasmid comprising the recombinant gene with a promoter and a terminator operably linked thereto so that the recombinant gene can be expressed in the cell; (c) transforming the cell with at least one recombinant plasmid prepared in step

(b); (d) expressing the reporter protein comprising the signal protein and the fluorescent protein in the transformed cell, while treating the transformed cell with a chemical before, after, or at the same time as the expression;

(e) detecting the cellular distribution of the reporter protein by monitoring the fluorescence image of the transformed cell treated with the chemical during the expression or trafficking, or thereafter; and

(f) determining the effect of the chemical by comparing the fluorescence image obtained in step (e) with that of a control transformed cell which is not treated with the chemical.

In the third aspect, the present invention provides recombinant genes encoding the reporter protems that are used in the above methods to visualize the trafficking of the reporter protems and their distributions in subcellular organelles. The recombinant gene comprises a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protein linked thereto. Details of the compositions of the present invention are described below. In the present invention, the signal protein is selected from the proteins that have trafficking signals targeting to nucleus, mitochondria, chloroplast, peroxisome, plasma membrane, endoplasmic reticulum, Golgi apparatus, storage vacuole, lytic vacuole, and prevacuolar compartment, and also those proteins targeting to 3 classes of phospolipids. Examples of the signal protems include NLS (nuclear localization sequence), AtOEP7, Cab (chlorophyll a/b binding protein), SKL (peroxisome targeting motif), RbcS (rubisco small subunit), RA (rubisco activase), Fl-H^-ATPase, ETVATPase, BiP (chaperone binding protein), ST (Sialyltransferase). Chi (chitinase), recombinant clone 526, clone 491, clone 500, AtVTIla, SPO (sporamin), EBD, AtPH, FAPP, PH, etc. The present invention provides methods for preparing transformed cells that can express reporter proteins, each comprising one of the signal proteins described above and a fluorescent protein label linked thereto. The present invention also provides a systematic method for selectively detecting the intracellular processes using the transformed cells and a systematic method for selectively screening drug candidates based on this detection method. More particularly, the methods of the present invention include a step of selecting a specific protein that has a property of translocating to a specific subcellular location, a step of synthesizing the whole gene of the selected signal protein or a portion thereof encoding the trafficking signal of the selected protein, a step of synthesizing a gene encoding a fluorescent protein that can be linked to the signal protem to fluorescently visualize the subcellular localization, and a step of constructing a recombinant gene comprising a gene encoding the signal protem and a gene encoding the fluorescent protein linked thereto. The function of the signal protem can change depending on the way that the fluorescent protein is linked to the signal protein. The present invention therefore provides compositions of the reporter proteins whose signal protems can correctly direct the trafficking, and also construction methods thereof.

The present invention also provides a procedure for constructing recombinant plasmids that can be used to express the recombinant genes in a cell. The recombinant plasmid can be constructed by ligating a recombinant gene into a vector containing a promoter, a terminator, and other necessary factors. As well known to the persons having ordinary skills in the art to which the present invention pertains, methods for transforming a cell by introducing the recombinant plasmid include, but are not limited to, chemical-mediated methods using PEG (polyethylene glycole), potassium phosphate, or DEAE-dextran, cationic lipid-mediated lipofection, microinjection, electroporation, and electrofusion. In the transformation, one type of the recombinant plasmid could be introduced, or else two or more types of the recombinant plasmids can be introduced to express two or more reporter protems simultaneously. The conditions need to be optimized to efficiently express the reporter protein comprising the signal protein and the fluorescent protein in the transformed cell. The persons skilled in the art can select appropriate conditions depending on the signal protem and the fluorescent protein used. Detailed structures of the recombinant plasmids are explained in the examples of the present invention to present the amino acid sequences of the reporter proteins or corresponding nucleic acid sequences.

In constructing the reporter proteins according to the present invention, the signal proteins were classified into three classes based on the protem trafficking mechanisms, in order to show that the methods of the present invention for constructing the reporter proteins can be commonly used for various mechanisms of protein trafficking to different subcellular organelles. Signal proteins targeting to nucleus, chloroplast, mitochondria, etc., correspond to the case that a specific portion of the signal protein directly acts as a recognition signal to direct the intracellular trafficking. These signal proteins are classified as Group I for convenience in the specification of the present invention (see Figure 1). Signal protems targeting to endoplasmic reticulum, Golgi apparatus, lytic vacuole, storage vacuole, plasma membrane, etc., correspond to the case of the endosomal trafficking in which a specific portion of the signal protein acts as a signal to be captured by endoplasmic reticulum so that the signal protein is translocated as enclosed in endoplasmic reticulum. These signal proteins are classified as Group II for convenience in the specification of the present invention (see Figure 4). In the present invention, detailed methods are provided for visualizing the trafficking processes and the cellular distributions of these classes of the signal proteins. In addition, signal proteins related to intracellular signal transduction via specific binding to phospolipids are selected in the present invention, and methods are provided for observing the subcellular organelles that contain specific phospholipid. These signal proteins are classified as Group III for convenience in the specifications of this invention (see Figure 6).

It will often be useful to practice the invention with reporter proteins having specific trafficking features. Thus for example in embodiments in which it is desirable to detect trafficking and localization of reporters with Group I or Group II signal proteins (or both Group I and Group II signal proteins), it will not be as useful to detect trafficking of report proteins having specific Group III signal protems.

Thus in an embodiment along these lines, the invention can be employed to detect trafficking and localization of a selected reporter protein in which the reporter includes a Group I or Group II signal protein. Preferably, such a reporter does not include a Group III signal protein as defined herein. In a more specific embodiment, the reporter protein of interest does not include the endosome binding domain (EBD) of human early endosome antigen 1 (a Group III signal protein) as defined by Kim D.H et al. (2001) in Plant Cell 13: 287 ie., amino acids 1257 to 1411 of EBD. In another embodiment, the reporter protem does not include one or more of the following signal proteins as defined in Figure 6: AfPH; FAAPl; and PH. In still another embodiment, the reporter protein for use with the present invention does not include a recognition site(s) that binds specifically to subcellular organelles that contain specific phospholipid as defined herein for the Group III family of signal protems.

The present invention thus provides a method for detecting trafficking and localization of a selected reporter protein inside a cell in which the protein does not include one or more of the EBD signal protem, AtPH; FAAPl; or PH as defined in Figure 6. Thus in this illustration of the invention, the method includes introducing and expressing the reporter protein inside cells and detecting the cellular distribution of that reporter to detect trafficking and localization of the desired protein within the cells. Such a method can also be used in accord with the invention as a screening platform to detect and optionally quantify trafficking activity of one or more compounds to be tested. Thus in one invention embodiment, the cell expressing the reporter protein (without one or more of the EBD, AtPH; FAAPl; or PH signal proteins as defined in Figure 6) can be contacted with at least one desired compound to be tested. The compound to be tested can be added to cultured cells before, during or after introduction of the reporter protein as discussed previously. Such a specific invention method is useful in a variety of settings eg., where detection of Group I, Group II, or both Group I and II trafficking is desired.

In other embodiments, it will often be useful to practice the invention methods with reporter proteins that do not include Group II signal protems. For instance, it will be useful to detect trafficking with reporters that do not include one or more of the following Group II signal proteins: ST; BiP; Sporamin; and H⁺-ATPase as defined by Kim D.H et al. (2001) in Plant Cell 13: 287. In other embodiments, it will be more useful to detect trafficking with reporters in which the signal protein does not include a recognition site(s) that is involved with endosomal trafficking such that a specific portion of the signal protein acts as a signal to be captured by endoplasmic reticulum. More particular signal protems along these lines, and which would not be as useful in this invention embodiment, are translocated as enclosed in endoplasmic reticulum to eg., the Golgi apparatus, lytic vacuole, storage vacuole, and plasma membrane. Accordingly, the invention provides a method for detecting trafficking and localization of a selected reporter protem inside a cell in which the protein does not include one or more of the ST; BiP; Sporamin; and H -ATPase signal proteins as defined eg., in Figure 4. In this illustration of the invention, the method includes introducing and expressing the reporter protein inside cells and detecting the cellular distribution of that reporter to detect trafficking and localization of the desired protein within the cells. Importantly, the method can also be used in accord with the invention as a screening platform to detect and optionally quantify trafficking activity of one or more compounds to be tested. Thus in one invention embodiment, the cell expressing the reporter protein (without one or more of the ST: BiP; Sporamin; and H⁺-ATPase signal protems as defined in Figure 4) can be contacted with at least one desired compound to be tested. The compound to be tested can be added to cultured cells before, during or after introduction of the reporter protein as discussed previously. Such a specific invention method has a variety of important uses such as in settings in which detection of Group I, Group III, or both Group I and III trafficking is desired.

Using the trafficking properties of these signal proteins, details of the subcellular localization of the signal protems can be specifically visualized, for example, on the membrane, or inside or outside of an organelle. The present invention also provides a method for visualizing the localization of two or more proteins simultaneously by using two or more fluorescent proteins with different colors.

In the embodiments of the present invention, green fluorescent protein (GFP, Davis and Viestra, 1998) and red fluorescent protein (RFP) are used for constructing the reporter protems to visualize their cellular localization. However, the person having ordinary skill in the art to which the present invention pertains can fully understand that the reporter proteins of the present invention can be constructed by using fluorescent proteins other than GFP and RFP. The expression and trafficking processes of the reporter protein can be visualized in details for each stage of the processes by continuously monitoring the images of the fluorescence emitted by the reporter protem expressed in the transformed cell, using a fluorescence microscope at a specific wavelength.

As it becomes possible to detect the expression and trafficking processes of a specific protein and its cellular distribution, a selective drug screening system for identifying chemicals inhibiting or enhancing the intracellular trafficking of the selected protein is established using this detection method. More particularly, it is demonstrated that chemicals affecting the intracellular trafficking can be identified by treating the transformed cell with a chemical before, after, or at the same time as the expression of the reporter protein, and then identifying the effect of the chemical by comparing the cellular distribution of the reporter protein in the transformed cell treated with the chemical with that in the control transformed cell which is not treated with the chemical. The same method can be used to screen chemicals inhibiting or enhancing the transcription or the translation of proteins because the level of the protein expression can be examined from decrease or increase in the intensity of the fluorescence signal from the reporter protein. This is also demonstrated in the examples of the present invention.

Morphological changes induced by a chemical, such as modification, damage, or destruction of the subcellular organelles can be detected by observing the distribution or pattern of the fluorescence signal from the reporter protein. Therefore, it is also possible to screen cytotoxic chemicals that cause alteration of the subcellular organelles. This is also demonstrated in the examples of the present invention.

In order to show that the selective drug screening system provided by the present invention can be used practically, known inhibitors such as bafilomycin Al, wortmannin, and brefeldin A are examined to confirm that inhibition of the intracellular trafficking and expression processes and also cytotoxicity causing morphological changes of the subcellular organelles can be detected practically. This is described in detail in the examples of the present invention. As described in the examples, the signal proteins included in Group I, II, and III are examined to check the difference arising from the protein trafficking mechanisms. Compositions and usages of the present invention are described in detail in the embodiments with the attached drawings. The embodiments are to explain, but not limit, the present invention. The person having ordinary skill in the art to which this invention pertains can easily recognize other objects and advantages of the present invention from the attached drawings, the detailed description, and the claims of the present invention.

The following Examples are illustrative of the present invention.

Example 1. Construction of recombinant plasmids for expression of Group I proteins targeting to the organelles across the membrane. The coding region for the transit peptide of Fl-H⁺- ATPase (access number

D88374) was amplified by polymerase chain reaction (PCR) from a λZAPII cDNA library using two specific primers (5'-CTTTAATCAATGGCAATG and 5'-

CCATGGCCTGAACTGCTCTAAGCTT) and ligated in-frame to the 5' end of the coding region of the green fluorescent protein to generate a recombinant gene for Fl- H⁺-ATPase:RFP (Niwa et al., 1999). The recombinant gene was subcloned into pUC under the control of the 35S promoter to construct a recombinant plasmid for ATPase:RFP. The same method was used for construction of other recombinant plasmids.

To express the reporter protein of the ribulose bisphosphate carboxylase (Rubisco) complex, the coding region for the transit peptide of the small subunit of the

Rubisco complex was PCR amplified from a λZAPII cDNA library using two specific primers (5'-CCTCAGTCACACAAAGAG and 5'-

ACTCGAGGGAATCGGTAAGGTCAG). The resulting PCR product was subcloned into pBluescript and subsequently ligated in-frame to the 5' end of the coding regions of GFP and RFP to construct recombinant plasmids for RbcS:GFP and RbcS:RFP, respectively.

The coding region of the chloroplast a/b binding protein was PCR amplified from a λZAPII cDNA library using two specific primers (5'-

TAGAGAGAAACGATGGCG and 5'-GGATCCCGTTTGGGAGTGGAACTCC) and used to construct a recombinant plasmid for Cab:GFP .

The coding regions of the transit peptide of rubisco activase (RA) was PCR amplified from a λZAPII cDNA library using two specific primers (5'-

TCTAGAATGGCCGCCGCAGTTTCC and 5'-

GGATCCATCTGTCTCCATCGGTTTG) and ligated to the 5' end of the coding regions of GFP and RFP to construct recombinant plasmids for RA:GFP and RA:RFP, respectively.

The coding region of the Arabidopsis outer envelope membrane protem,

AtOEP7, a homolog of OEP14 of pea was PCR amplified from a Arabidopsis genomic

DNA using two specific primers (OEP7-F: 5'-GACGACGACGCAGCGATG and OEP7-R: 5'-GGATCCCCAAACCCTCTTTGGATGT) which were designed to remove the natural termination codon, and subsequently ligated to the 5' end of the coding regions of GFP and RFP to construct recombinant plasmids for AtOEP7:GFP and

AtOEP:RFP, respectively.

A recombinant plasmid for the nuclear localization signal (NLS), NLS:GFP, was constructed as described previously (Pih et al., 2000). A recombinant plasmid for

NLS:RFP was constructed by replacing the GFP coding region with the RFP coding region in the recombinant gene for NLS:GFP.

The recombinant plasmid for the peroxisomal reporter GFP:SKL (serine, lysine, leucine) was constructed by PCR with 326GFP (Davis and Viestra, 1998) as a template using two specific primers (5'-CCGTATGTTACATCACC and 5'- TTATAGCTTTGATTTGTATAGTTCATCCAT).

Schematic diagrams of the reporter proteins constructed by the methods described above are presented in Figure 1.

Example 2. Preparation of protoplasts and transformation of protoplasts with recombinant plasmids.

(a) Preparation of protoplasts. Leaf tissues (5g) of 3-4 week-old Arabidopsis plants grown on soil in a green house were cut into small squares (5-10 mm²) with a new razor blade and incubated with 50 ml of the enzyme solution (0.25% Macerozyme R-10, 1.0% Cellulase R-10, 400 mM mannitol, 8 mM CaCl₂, 5 mM Mes-KOH, pH 5.6) at 22°C with gentle agitation

(50-75rpm). After incubation, the protoplast suspension was filtered through a 100 μm mesh and protoplasts were collected by centrifugation at 46xg for 5 min. The pelleted protoplasts were resuspended in 5 to 10 ml of the W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KC1, 5 mM glucose, 1.5 mM Mes-KOH, pH 5.6), overlaid on top of 20 ml of 21% sucrose, and centrifuged for 10 min. at 78xg. The intact protoplasts at the interface were transferred to a new tube containing 20 ml of the W5 solution. The protoplasts were pelleted again by centrifugation at 55xg for 5 min and resuspended in 20 ml of the W5 solution. The protoplasts were incubated on ice for 30 minutes.

(b) Isolation of recombinant plasmid DNAs and transformation of protoplasts. Recombinant plasmids were purified using Qiagen columns (Valencia, CA) according to the manufacture's protocol. To transform the protoplasts with the DNA, the protoplasts were pelleted again by centrifuge at 46xg for 5 min and resuspended in the MaMg solution (400 mM Mannitol, 15 mM MgCl₂, 5 mM Mes-KOH, pH 5.6) at a density of 5x10⁶ protoplasts/ml. The recombinant plasmids were introduced into Arabidodsis protoplasts by PEG-mediated transformation (Kim et al., 2001; Jin et al., 2001). Plasmid DNA (about 20-50 μg at a concentration of 2 μg/μl) was added to 300 μl of the protoplast suspension, and subsequently 325 μl of the PEG solution (400 mM Mannitol, 100 mM Ca(N0₃)₂, 40% PEG 4000) was added. The mixture was gently mixed and incubated for 30 min at room temperature. After incubation, the mixture was diluted with 10 ml of the W5 solution. Protoplasts were recovered by centrifugation at 50xg for 5 min and resuspended in 3 ml of the W5 solution and incubated at 22°C in the dark.

Example 3. Expression of Group I reporter proteins and observation of their expression and localization. The recombinant plasmids constructed in Example 1 were used to transform the protoplasts according to the method described in Example 2. The expression of the reporter proteins after the transformation was monitored as a function of time by capturing images using a fluorescence microscope (Axioplan fluorescence microscope, Zeiss, Germany) equipped with a cooled charge-coupled device (CCD) camera. The filter sets used were XF116 (exciter: 474AF20, dichroic: 500DRLP, emitter: 510AF23), XF33/E (exciter: 535DF35, dichroic: 570DRLP; emitter, 605DF50), and XF137 (exciter, 540AF30; dichroic, 570DRLP, emitter: 585ALP) (Omega, Inc, Brattleboro, VT) for GFP, RFP, and auto-fluorescence of chlorophyll, respectively. Data were then processed using Adobe (Mountain View, CA) Photoshop software, and the images were rendered in pseudo-color.

(a) Localization of the chloroplast targeting reporter protems.

The green fluorescence of the reporter protein AtOEP7:GFP was observed at the outer envelop membrane of the chloroplast. The red fluorescence in Figure 2a is the auto-fluorescence of chloroplasts. Figure 2b shows the image obtained by eliminating this auto-fluorescence by using a filter. This result indicates that the chimeric protein comprising the signal protein with the chloroplast envelope targeting signal and the fluorescent protein label was correctly targeted to the chloroplast envelope membrane.

Localization of the green fluorescence of the chimeric proteins, RbcS:GFP, Cab:GFP, and RA:GFP are presented in Figure 2(c), 2(d), and 2(e), respectively. As shown in the figures, RbcS:GFP was targeted to the stroma of chloroplast, and Cab:GFP and RA:GFP also emitted the fluorescence in the chloroplast. These results indicate that the chimeric protems comprising the trafficking signal of RbcS, Cab, or RA and fluorescent reporter protein were targeted to the chloroplast.

(b) Mitochondria targeting of Fl-H⁺-ATPase:RFP.

The red fluorescence of the reporter protein Fl-H⁺-ATPase:RFP was observed in the mitochondria (Figure 2(f)). This result indicates the chimeric protein comprising the signal protem with the mitochondria targeting signal and the fluorescent protein was transported into the mitochondria. (c) Peroxisome targeting of GFP:SKL.

By analyzing the location of the green fluorescence from the reporter protein

GFP:SKL, it was observed that the green fluorescence of GFP:SKL was transported to the peroxisome as shown in Figure 2(g). This result indicates that the reporter protein comprising the peroxisome targeting signal, SKL (serine, lysine, leucine), and the fluorescent protein label was translocated to the peroxisome.

(d) Nuclear targeting of NLS:GFP.

By analyzing the location of the green fluorescence from the reporter protem, it was observed that the green fluorescence of NLS:GFP was transported to the nucleus as shown in Figure 2(h). This result indicates that the reporter protein comprising the nuclear localization signal and the fluorescent protein label was translocated to the nucleus.

Example 4. Confirmation of the chloroplast envelope targeting of

AtOEP7:GFP by Western blot analysis.

The recombinant plasmid for AtOEP7:GFP was constructed according to the method in Example 1. This recombinant plasmid was used to transform protoplasts according to the method in Example 2, and the transformed protoplasts were incubated for 24 hrs at 22°C. The total protein extract was prepared as follows. Five ml of cell lysate was centrifuged, suspended in 5 ml of the extraction solution (10 mM EDTA, 50 mM HEPES-KOH, 0.33 M sorbitol, 0.5 g/1 BSA, 5 mM sodium ascorbate) at 4°C, and homogenized every three seconds for 20 min. The total protein extract was fractionated by ultra-centrifugation at 100,000xg to separate the soluble and membrane fractions. Both fractions were then electrophoresed on a 7.5% SDS/PAGE gel and transferred onto the PVDF membrane. The blot was probed with a polyclonal anti-GFP antibody.

As presented in Figure 3, the result shows that the expressed signal protein was transported to the chloroplast envelope membrane and not present in the cytosol. This result indicates that localization of proteins, which is conventionally determined by Western blot analysis, can be identified by the method provided by the present invention.

Example 5. Construction of recombinant plasmids for expression of Group II protems that are transported to subcellular organelles by endosomal trafficking.

The full length coding sequence of H⁺- ATPase (Arabidopsis AHA2) was amplified with two specific primers (5'-GAGATGTCGAGTCTCGAA and 5'-

CTCGAGCACAGTGTAGTGACTGG) and ligated to the 5' end of the GFP coding sequence. The ligated recombinant gene was subcloned into the pUC vector under the control of the 35S promoter to construct a recombinant plasmid for H⁺-ATPase:GFP. The same procedure was applied in the following examples.

The coding sequence of the chaperone binding protein (BiP) (access number D82817) was amplified from an Arabidodsis cDNA library using two specific primers, BIP5 (5'-TACGCAAAAGTTTCCGAT-3') and BIP3 (5'-

CTAGAGCTCATCGTGAGA-3'). The amino terminal region (44 amino acids) and the carboxyl terminal region (80 amino acids) of this gene were ligated to the amino terminus and the carboxyl terminus of GFP or RFP, respectively, to construct recombinant plasmids for BiP:GFP and BiP:RFP.

The sialtransferase (ST) cDNA was amplified from a λZAPII cDNA library using two specific primers (5'-ATGATTCATACCAACTTGAAG and 5'- GGATCCACAACGAATGTTCCGGAA). GFP or RFP was ligated in-frame to the carboxyl terminus of ST to construct ST:GFP or ST:RFP. To construct a recombinant plasmid for the chimeric protem Chi-n:RFP:Chi-c, a

DNA fragment including the RFP coding sequence without the termination codon was inserted into the Sma I and Eco RV sites of the chitinase cDNA of pea (access number M13968).

To express clone 500 encoding the vacuolar sorting receptor protem (BP-80) without the cytoplasmic tail, a recombinant plasmid for 500:GFP was constructed by inserting the GFP coding region without the termination codon into the EcoRI site of clone 500 (Kim et al., 2001).

For clone 526 encoding BP-80 with its cytoplasmic tail substituted by the tonoplast intrinsic protein (TIP), a recombinant plasmid for 526:GFP was constructed by inserting the coding region of GFP into the EcoRI site of clone 526.

To express the reporter protem for clone 491 encoding the BP-80 protem, recombinant plasmids for 491:GFP and 491:RFP were constructed by inserting clone 491 into the 5' end of the coding regions of GFP and RFP without the termination codon, respectively. A recombinant plasmid for 500:GFP:KKXX was generated as follows: The

GFP coding region without the termination codon was inserted into the EcoRI site of clone 500 (Jiang and Rogers, 1998) and KKXX was then added to the C-terminus of 500:GFP by PCR amplification using two specific primers (5'- GGATCCTCTAGAGGATCGATCCGG and 5'- TTAGATGAGTTTCTTTTTCTCAAAGAAAGTTTTCAAAAGGAATCCCCCTCC). To express AtVTIla, a homolog of Arabidopsis t-SNARE which is transported from the trans-Golgi network to the storage prevacuole (Zheng et al., 1999), a recombinant plasmid for RFP: AtVTIla was constructed by ligating the coding region of AtVTIla to the C-terminus of the RFP coding region. A recombinant plasmid for AtVTIl :GFP was constructed by ligating the coding region of GFP to the C-terminus of the coding region of AtVTIla.

To express sporamin, a recombinant plasmid for SPO:GFP was constructed by ligating GFP to the carboxyl terminus of the sporamin B gene.

Schematic diagrams of the reporter proteins expressed from the recombinant plasmids constructed as above are shown in Figure 4.

Example 6. Observation of the expression and localization of Group II reporter proteins.

Recombinant plasmids for H⁺-ATPase:GFP, ST:GFP, BiP:GFP, 526:GFP, Chi- n:RFP:Chi-c, and 500:GFP:KKXX were constructed as described in Example 5 and used to transform the protoplasts by the method of Example 2. Expression of the reporter proteins was monitored as a function of time using a fluorescence microscope as explained in Example 3. A part of the results is given in the following.

Fluorescence of the reporter protein H⁺-ATPase:GFP was observed in the plasma membrane (Figure 5(a)). Fluorescence of ST: GFP was observed in the Golgi apparatus (Figure 5(b)). The red fluorescence in these images is the auto-fluorescence of chloroplasts. Reporter proteins, BiP:RFP and 500:GFP:KKXX showed fluorescence in the lumen and the membrane of the endoplasmic reticulum, respectively (Figure 5(c) and 5(d)). 526:GFP showed fluorescence on the membrane of the storage vacuole (Figure 5(e)) and Chi-n:RFP:Chi-c showed fluorescence in the storage vacuole (Figure 5(f)). When chitinase was used as a signal protem, Chi-n:RFP, in which the carboxyl region of chitinase was not ligated, was not targeted to the storage vacuole, but it was present as speckles in the endoplasmic reticulum (Figure 5(g)). Fluorescence of SPO:GFP was distributed uniformly throughout the lytic vacuole (Figure 5(h)).

Example 7. Construction of recombinant plasmids to express Group III proteins that are specific to phospholipids.

To construct the recombinant DNA for GFP:EBD, the C-terminal coding region (amino acid residue 1257 to 1411) of human early endosome antigen 1 (EEA1) was PCR amplified with two primers 5'-GAATTCGTGGCAATCTAGTCAACGG-3' and 5'-CTAATGTTAGTGTAATATTAC-3', and ligated to the C-terminus of the GFP coding sequence without the termination codon. This recombinant DNA was inserted to a pUC vector under the control of the 35S promoter to construct a recombinant plasmid. The same cloning procedure was applied in the examples hereafter.

A recombinant plasmid for a EBD derivative, GFP:EBDC1358S, was prepared using a primer directing replacement of the amino acid residue 1358 to serine.

A recombinant plasmid for the chimeric protein of Arabidopsis Pleckstrin homology (PH) domain, GFP:AtPH, was constructed by PCR amplification using two primers 5'-CCCGGGAAATGGAGAGTATGTGGCGA-3' and 5'-

TAATCACCGCCTGTGATCATA-3'. A recombinant plasmid for the chimeric protein of FAPP including the PH domain, GFP:FAPP, was constructed by PCR amplification using two primers 5'-CTCGAGATGGAGGGGGTTCTGTACAAG-3' and 5'- TCACGCTTTGGAGCTCCCAAGGGC-3'. A recombinant plasmid for PH:GFP was constructed by the method of Kost B et al. (1998).

Schematic diagrams of the reporter proteins constructed as above are shown in Figure 6.

Example 8. Observation of the expression and localization of Group III reporter proteins.

Recombinant plasmids for GFP:EBD, GFP:AtPH, GFP:FAPP, and GFP:PH, were constructed as described in Example 7 and used to transform the protoplasts by the method of Example 2. Expression of the reporter proteins was monitored as a function of time using a fluorescence microscope as explained in Example 3. A part of the results is given in the following.

The reporter proteins, GFP:EBD and GFP: AtPH, showed fluorescence at the outer membrane of vacuole, indicating the presence of phosphatidylmositol 3 -phosphate (PI(3)P) in the outer membrane of vacuole. GFP:FAPP and GFP:PH showed fluorescence at the plasma membrane indicating the presence of phosphatidylmositol 4- phosphate (PI(4)P) and phosphatidylmositol 4,5-diphosphate (PI(4,5)P2), respectively, in the plasma membrane. These results show that it is possible to use the phospholipid- specific protein or its portion as a signal protein to target a protein to the phospholipid- containing cellular compartments (see Figure 7).

Example 9. The effect of wortmannin on the intracellular trafficking of RbcS:GFP. (a) Plasmid construction, transformation, and expression of the chimeric protem. A recombinant plasmid for RbcS:GFP was constructed as described in Example 1. Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2. The protoplast suspension was treated with wortmannin at a concentration of 5 μg/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the chimeric protem was observed as in Example 3.

(b) The effect of wortmannin on the intracellular trafficking of the chimeric protein. The effect of wortmannin on the trafficking of RbcS:GFP was examined.

Wortmannin is known as a specific inhibitor of phosphatidyl 3 -phosphate (PI(3)P) and phosphatidylmositol 4-phosρhate (PI(4)P) (Ui et al., 1995). Localization of the green fluorescence in the wortmannin-treated protoplast was compared with that of the control protoplast that was not treated with wortmannin. As shown in Figure 8, in contrast to the control protoplast in which the green fluorescence of RbcS:GFP was targeted to the chloroplast as expected (Figure 8(a)), the green fluorescence was not translocated to the chloroplast in the presence of wortmannin, but observed as either speckles or aggregates (Figure 8(b) and 8(c)). This result indicates that wortmannin inhibits trafficking of the chloroplast-targeting protem from the cytosol to the chloroplast.

Example 10. The effect of bafilomycin Al (BafAl), known as an inhibitor of the vacuolar type H -ATPase, on retrograde trafficking of 500:GFP:KKXX.

(a) Plasmid construction, transformation, and expression of the chimeric protem. The recombinant plasmid for 500:GFP:KKXX was constructed as described in Example 5. Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2. The protoplast suspension was treated with bafilomycin Al at a concentration of 5 μg/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the chimeric protem was observed as in Example 3.

(b) The effect of bafilomycin Al (BafAl) on the trafficking of the chimeric protem.

Localization of the green fluorescence in the BafAl -treated protoplast was compared with that of the control protoplast that was not treated with BafAl. As shown in Figure 9, while the green fluorescence was observed as numerous networks in the control protoplast (Figure 9(a) and 9(b)), it was observed as a ring pattern on the plasma membrane (Figure 9c) and the vacuolar membranes (Figure 9d) in the presence of BafAl. This result indicates that BafAl inhibits the retrograde trafficking of 500:GFP:KKXX in the Arabidopsis protoplast and causes transport of the reporter protein to the plasma membrane or the vacuolar membrane.

Example 11. The effect of a specific inhibitor on the localization of two reporter proteins.

(a) Plasmid construction, transformation, and expression of the chimeric protem Recombinant plasmids for the reporter proteins, BiP:GFP and ST:RFP that are specific to the endoplasmic reticulum and the Golgi apparatus, respectively, were constructed as described in Example 5. Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2. The protoplast suspension was treated with brefeldin A at a concentration of 5 μg/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the chimeric protein was observed as in Example 3.

(b) Distribution of fluorescence from the two reporter proteins.

Brefeldin A (BFA) is known as an inhibitor of ADP-ribosylation factors (Arfs) in the animal cell (Morinaga et al., 1999). In the control protoplast, the green fluorescence of BiP:GFP was observed in the endoplasmic reticulum (Figure 10(a)) and the red fluorescence of ST:RFP was observed in the Golgi apparatus (Figure 10(b)). When the protoplast was treated with BFA, ST: GFP was targeted to the endoplasmic reticulum instead of the Golgi apparatus that was destroyed by BFA (Figure 10(e)). As observed, the fluorescent images of the chemical-treated protoplasts are distinctively different from those of the control protoplasts (Figure 10(c) and 10(f)). This suggests that the intracellular trafficking of two or more protems can be simultaneously detected by using two or more fluorescent proteins with different colors. The result observed for ST:GFP shows the destruction process of the Golgi apparatus.

Example 12. The effect of brefeldin A (BFA) on the biogenesis and structure conservation of the endoplasmic reticulum.

(a) Plasmid construction, transformation, and expression of the chimeric protein.

A recombinant plasmid for BiP:RFP was constructed as described in Example 5.

Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2. The protoplast suspension was treated with brefeldin A at a concentration of 5 μg/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the chimeric protein was observed as in Example 3.

(b) The effect of chemicals on the trafficking of the chimeric protein.

Localization of the red fluorescence in the BFA-treated protoplast was compared with that of the control protoplast that was not treated with BFA. As shown in

Figure 11(a), while the red fluorescence of BiP:RFP was present along the structure of the endoplasmic reticulum in the control protoplast, the structure of the ER was observed to be destroyed (Figure 11(c)) when treated with BFA. Effect of using a reporter protein that specifically binds to a subcellular organelle can be clarified by comparing the fluorescence images with the images taken by an optical microscope under bright field (Figure 11(a) and 11(d)).

Examplel3. Inhibition of trafficking of proteins that are specific to phospholipids. (a) Plasmid construction, transformation, and expression of the chimeric protein.

Recombinant plasmids for the reporter proteins, GFP:EBD and

GFP:EBDC1 58S were constructed as described in Example 7. These recombinant plasmids were used to transform the protoplasts according to the method of Example 2.

The transformed protoplasts were treated with wortmannin at a concentration of 1.0 μg/ml or with 2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4-on, a specific inhibitor of phosphatidylmositol 3 -kinase, at a concentration of 10 μg/ml (LY294002, Vlahos et al., 1994) and incubated at 22°C. Fluorescence images were monitored at various time points.

(b) The effect of chemicals on the localization of the chimeric proteins.

In the control protoplast that was not treated with the chemicals, the green fluorescence was localized in the endosome along the distribution of the EBD-binding phospholipid PI(3)P (Figure 12(a)). In the protoplasts treated with wortmannin or LY294002, however, the green fluorescence was distributed uniformly throughout the cytosol (Figure 12(a)). On the other hand, when the same experiment was performed for GFP:EBDC1358S in which the amino acid residue 1358 is mutates, the chemicals did not affect the distribution of the fluorescence.

Example 14. Change in the expression level induced by cycloheximide, an inhibitor of the protein expression.

(a) Plasmid construction, transformation, and expression of the chimeric protein. A recombinant palsmid for RA:GFP was constructed as described in Example 1. Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2. The protoplast suspension was treated with cycloheximide a concentration of 5 μg/ml. Then the protoplasts were transformed and incubated at 22°C in the dark. Expression of the chimeric protein was observed as in Example 3.

(b) The effect of the chemical on the expression of the chimeric protein.

The intensity of the green fluorescence was decreased in the cycloheximide treated-protoplast (Figure 13(a)) compared to that in the control protoplast that was not treated with cycloheximide (Figure 13(b)). This result shows that the expression of the reporter protein is inhibited, suggesting that the effect of a chemical on the transcription and/or translation of protein can be visualized. The red fluorescence images in Figure

13(c) and 13(d) shows the auto-fluorescence of the chloroplast, which are measured to relatively compare the growth and metabolism of the cells.

The present invention described above is not limited by the aforementioned examples and the attached drawings. The present invention can be substituted, changed, and modified without departing from the technical thoughts described in the specification and the claims, and such substitutions, changes, and modifications fall within the spirit and scope of the present invention.

All references disclosed in this application are incorporated by reference. The following references are specifically incoφorated by reference.

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Claims

What is claimed is:

1. A method for detecting trafficking and localization of a selected protein in a cell, which method comprises:

(a) preparing a recombinant gene comprising a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protem linked thereto;

(b) preparing a recombinant plasmid including the recombinant gene with a promoter and a terminator operably linked thereto so that the recombinant gene can be expressed in the cell;

(c) transforming the cell with at least one recombinant plasmid prepared in step (b);

2. A method for screening chemicals that affect specific characteristics related to trafficking and localization of a selected protem in a cell, which method comprises:

(a) preparing a recombinant gene comprising a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protem linked thereto; (b) preparing a recombinant plasmid comprising the recombinant gene with a promoter and a terminator operably linked thereto so that the recombinant gene can be expressed in the cell (c) transforming the cell with at least one recombinant plasmid prepared in step (b), (d) expressing the reporter protem comprising the signal protein and the fluorescent protein in the transformed cell, while treating the transformed cell with a chemical before, after, or at the same time as the expression;

3. A method for detecting specific characteristics related to trafficking and localization of a selected protein in a plant cell, which method comprises:

(a) preparing a recombinant gene comprising a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protein linked thereto;

(c) transforming the protoplast of the plant cell whose cell wall is removed with at least one recombinant plasmid prepared in step (b);

(d) expressing the reporter protem comprising the signal protein and the fluorescent protein in the transformed cell; and

4. A method for screening chemicals that affect specific characteristics related to trafficking and localization of a selected protein in a plant cell, which method comprises:

(b) preparing a recombinant plasmid comprising the recombinant gene with a promoter and a terminator operably linked thereto so that the recombinant gene can be expressed in the cell;

(d) expressing the reporter protem comprising the signal protem and the fluorescent protein in the transformed cell, while treating the transformed cell with a chemical before, after, or at the same time as the expression;

(e) detecting the cellular distribution of the reporter protein by monitoring the fluorescence image of the transformed cell treated with the chemical during the expression or trafficking, or thereafter; and (f) determining the effect of the chemical by comparing the fluorescence image obtained in step (e) with that of a control transformed cell which is not treated with the chemical.

5. The method according to any of claims 1 and 2, wherein the cell is a eukaryotic cell.

6. The method according to any of claims 1 to 4, wherein the fluorescent protein is one selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), mutants thereof, and derivatives thereof.

7. The method according to any of claims 1 to 4, wherein the specific subcellular organelle targeted by the reporter protem is one selected from the group consisting of nucleus, mitochondria, chloroplast, and peroxisome.

8. The method according to any of claims 1 to 4, wherein the coding region of the signal protem included in the recombinant gene is a coding sequence of a full length protein selected from the group consisting of NLS (nuclear localization signal), AtOEP7, Cab (chlorophyll a/b binding protein), SKL (peroxisome targeting motif), RbcS (Rubisco Small Subunit), RA (Rubisco Activase), and Fl-H⁺- ATPase, or a portion thereof that includes the trafficking signal.

9. The method according to any of claims 1 to 4, wherein the specific subcellular organelle targeted by the reporter protein is one selected from the group consisting of plasma membrane, endoplasmic reticulum, Golgi apparatus, ribosome, lysosome, cytoskeleton, centriole, storage vacuole, lytic vacuole, and prevacuolar compartment.

10. The method according to any of claims 1 to 4, wherein the coding region of the signal protein included in the recombinant gene is a coding sequence of a full length protem selected from the group consisting of H⁺-ATPase, BiP (chaperon binding protein), ST (sialytransferase), Chi (chitinase), clone 526, clone 491, clone 500, AtVTIla, and SPO (sporamin), or a portion thereof that includes the trafficking signal.

11. The method according to any of claims 1 to 4, wherein the coding region of the signal protein included in the recombinant gene encodes a protein that binds specifically to a phospholipid.

12. The method according to any of claims 1 to 4, wherein the coding region of the signal protein included in the recombinant gene is a coding sequence of a full length protein selected from the group consisting of EBD, AtPH, FAPP1, and PH which bind specifically to phospholipids, or a portion thereof that includes the trafficking signal.

13. The method according to any of claims 2 and 4, wherein the chemical is one selected from the group consisting of chemical compounds, polypeptides, mixtures of chemical compounds and polypeptides, and extracts of natural products.

14. The method according to any of claims 1 to 4, wherein the fluorescence image is monitored at one or more fluorescence wavelengths using a fluorescence microscope equipped with optical filters, each of which transmit fluorescence at a specific wavelength.

15. The method according to any of claims 2 and 4, wherein the chemical inhibits or enhances the trafficking of the reporter protem, so that the trafficking speed or the distribution of the reporter protein in the transformed cell treated with the chemical is altered compared to those in the control transformed cell.

16. The method according to any of claims 2 and 4, wherem the chemical inhibits or enhances the transcription in which mRNA is produced from the recombinant plasmid or the translation in which the reporter gene is produced from mRNA, so that the intensity and the distribution of the reporter protein in the transformed cell treated with the chemical is altered compared to those in the control transformed cell.

17. The method according to claims 2 and 4, wherem the chemical causes the cytotoxicity such as deformation, damage, or disruption of the cell or the subcellular organelles, so that the intensity and the distribution of the reporter protein in the transformed cell treated with the chemical is altered compared to those in the control transformed cell.

18. The method according to any of claims 1 to 4, wherem the method of introducing the recombinant plasmid into the cell is one selected from the group consisting of the methods of using a chemical such as PEG (polyethylene glycol), calcium phosphate, and DEAE-dextran, lifofection using cationic lipid, microinjection, electroporation, and electrofusion.

19. The method according to any of claims 1 to 4, wherein introduction of the recombinant plasmid into the cell is conducted by using 5 to 40% PEG.

20. The method according to any of claims 1 to 4, wherem observation of the expression, the trafficking, or the localization of the reporter protein is conducted in the time range of 5 min to 80 hrs during or after the incubation of the transformed cell.

21. A recombinant gene comprising a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protein linked thereto.

22. The recombinant gene according to claim 21, wherein the coding region of the signal protem is a coding sequence of a full length protein selected from the group consisting of AtOEP7 (Arabidopsis outer membrane envelope), RbcS (Rubisco small subunit), RA (Rubisco activase), Cab (Chlorophyll a b binding protein), H⁺-ATPase, BiP (Chaperon binding protem), Chi (Chitinase), clone 526, clone 491, clone 500, AtVTIla, SPO (sporamin), AtPH, and FAPP1, or a portion thereof that includes the trafficking signal.

23. The recombinant gene according to claim 21, wherem the specific subcellular organelle targeted by the reporter gene expressed from the recombinant gene is one selected from the group consisting of chloroplast, plasma membrane, endoplasmic reticulum, Golgi apparatus, storage vacuole, lytic vacuole, and prevacuolar compartment.

24. The recombinant gene according to claim 21, wherein the fluorescent protein is one selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), mutants thereof, and derivatives thereof.

25. A recombinant plasmid comprising the recombinant gene of any of claims 21 to 24 with a promoter and a terminator operably linked thereto so that the recombinant gene can be expressed in a cell.

26 A chimeric protein expressed from the recombinant gene of any of claims 21 to 24.

27. A method of using the chimeric protein of claim 26 as a carrier for targeting chemicals to a subcellular organelle.

28. The recombinant plasmid of claim 25, wherein the promoter is 35S promoter.

29. A method for detecting trafficking of a selected protein in a cell, which method comprises expressing in the cell at least one reporter protein comprising at least one signal protein and at least one fluorescent tag and detecting the reporter protein to detect trafficking of the selected protem in the cell.

30. A method for detecting a compound that modulates trafficking of a selected protem in a cell, the method comprising expressing in the cell at least one reporter protein comprising at least one signal protem and at least one fluorescent tag, contacting the cell with the compound under conditions conducive to modulating protein trafficking in the cell and detecting the reporter protein to detect the compound.

31. The method of any one of claims 1-4, 29 and 30, wherem the reporter protein does not include a Group III signal protein.

32. The method of claim 31, wherein the Group III signal protein excluded from the reporter protein is an endosome binding domain (EBD) of human early endosome antigen 1.

33. The method of claim 32, wherem the EBD domain consists of amino acids 1257 to 1411 of the human early endosome antigen 1.

34. The method of claims 1-4 and 29-33, wherein the reporter protein does not include a Group II signal protein.

35. The method of claim 34, wherein the Group II signal protein excluded from the reporter protein is one of ST; BiP; Sporamin; and H⁺-ATPase.