Below, you will find five questions having to do with material covered in lecture by Dr. Dean. You will choose to deal with only 2 of these. These questions are hypotheses that may or may not be true. To our knowledge, none of these questions has been experimentally addressed to date. Your task is to design a set of experiments, with all necessary controls, to test these hypotheses. You need to assume that all aspects of the statement require proof (except for obvious ones such as "macrophage lysosomes" - lysosomes can be assumed to be present in macrophages as they are in all other mammalian cells). Try and use two or more approaches to address a given problem, e.g., a biochemical experiment coupled with a a microscopic experiment; the results obtained will be much more convincing.

You have until 5 p.m. on Feb. 2, 1998 to design and write a maximum of 1 typewritten page per hypothesis (0.5 in borders all around, font size 10 or better). Please remember to write your name on each sheet. You may use whatever library facilities you need. In contrast to past in-class exams of this nature, it is expected that the experiments proposed in response to these questions be capable of being performed in a reasonable length of time and to address the hypothesis as completely as possible.

1. Does phosphatidylinositol-4,5-bisphosphate cause an increase in the activity of phospholipase D, when bound to myristoylated ARF1p, that results in an increase of COPI binding to Golgi-derived vesicles?
1. Do macrophage lysosomes behave like regulated secretory vesicles because they interact with the rab3A protein?
1. Do M. tuberculosis-containing vacuoles not fuse with lysosomal enzyme-containing vesicles because they (the vacuoles) lack ARF5?
1. Can the import of tRNA into mitochondria from the cytosol be driven by a D pH?
1. Does transportin function as a nuclear export signal (NES) receptor, in addition to functioning as a nuclear localization sequence (NLS) receptor?

QUESTION #1: Phosphatidylinositol-4,5-bisphosphate (PIP2) causes an increase in the activity of phospholipase D, when bound to myristoylated ARF I p, that results in an increase of COP I binding to Golgi-derived vesicles. (CJ)

MATERIALS:

a. phospholipids: PE (phosphatidyl ethanolamine), PIP2, PC (dipalmitoyl phosphatidylcholine), PIP (phosphatidylinositol-4phosphate), PS (phosphatidylserine)

4. bovine brain cytosol - separate coatomer from ARF by gel filtration of cytosol. elute with KCI-Tris pH 7.8. collect fractions. analyze by SDS-PAGE and immunoblotting with monoclonal antibodies to b -COP and ARF5. Brefeldin A.

EXPERIMENTAL APPROACH Although it is known that ARFlp interacts with PLD in cells, the mechanism of interaction is not entirely understood. It has been demonstrated that myristolylated ARF I stimulates PLD activity (Brown, et al. 1993). In addition, PEP2 is required for optimal stimulation of PLD activity m comparison to PIP and PS (Brown, et al. 1993). However, whether the interactions between ARF, PLD and PEP2 play a more important role in signal transduction or membrane trafficking has yet to be detected. Both experiments are designed to assess if phosphoinositides contribute to the regulation of ARF I p binding to vesicles and consequently the recruitment of coatomer binding to vesicles. Experiment I investigates the effect of three different phosphoinositides (PS., PIP, PIP2) on ARF I p binding to coatomers versus their effect on coatomer binding and PLD as it is fused to ARF I p. Coatomer and ARFlp are detected as mg monoclonal antibodies to b -COP (subunit of coatomer) and ARFlp, respectively. Experiment 2 more specifically measures PLD activity by measuring the amount of hydrolyzed product. Brefeldin A serves as control, as it is a protein inhibitor and acts to prevent the assembly of ARF-coatomer complexes.

EXP’T 1: B-COP/ARF BINDING ASSAY

EXP’T 2: ASSAY OF PLD ACTIVITY BY HYDROLYSIS OF PC

4. Centrifuge and remove aliquot of supernatant and analyze by scintillation counter.

Northern blot analysis - Lyse macrophages, collect mRNA, and separate by agarose gel electrophoresis. Transfer to a nitrocellulose membrane and expose to a radioactively labeled rab3A probe. Subject to autoradiography.

Electron microscopy - Use a detergent to permeabilize the membrane. Incubate with anti rab3A Ab tagged with large gold particles and with an Ab to acid phosphatase (lysosomal enzyme) tagged with small gold particles. If rab3A interacts with macrophage lysosomes, should see small gold particles inside the lysosome and large gold particles at the surrounding membrane.

Previous work: proteins stored inside the cell (regulated secretary vesicles) are characterized by a core of aggregated protein - most mammalian cells contain chromogranin B, p38, and secretogranin 11 that @, in aggregates that appear as dense cores in vesicles on EM micrographs. f I;

a. Strip probe off nitrocellulose membrane used in question 1. Use a probe for chromogranin B (or other protein known to form aggregates in regulated secretary vesicles). Do autoradiography. (If negative, repeat until find protein in lysosome granules known to be localized in regulated secretary vesicles).

b. Western blot - Lyse macrophages, use differential centrifugation to collect vesicles, use a detergent to disrupt the vesicles, and run on SDS-PAGE. As a control, include regulated secretary vesicles (insulin) and constitutive vesicles that continually release their contents (albumin). Transfer to a nitrocellulose membrane and use an antichromogranin Ab and autoradiograph. The Ab should bind to all regulated secretary vesicles but should not bind to albumin.

c. Permeabilize macrophages with detergent and add anti-chromogranin B Ab tagged with FITC. Examine with a confocal fluorescent microscope. Should see lysosomes fluorescing in cytosol but should not see any fluorescence being released into the extracellular matrix.

Create a mutant rab3A that functions in a dominant negative fashion. (Or select a ts mutant rab3a expressed in a rab3a-knock-out mouse.) Repeat the assays to determine if protein aggregates form in the vesicles with the dominant neg rab3a (or better, using the ts rab3a at both permissive and restrictive temperatures). Compare EM micrographs to those above to see if the vesicles continue to act like regulated secretary vesicles. Since a rab3A knockout mouse already exists (Sudhof, 1995), collect macrophages from the mouse and use the same assays (the FITC tag will show differences in pH - if the mutant rab3A blocks binding or disrupts protein aggregation, a change in the pH of the vesicles may be observed). A disruption of rab3A may also cause the lysosomal vesicles to be secreted instead of being stored inside the cell. To test, collect macrophages from the knockout, incubate with ³H Leu, wash with cold aa precursors, and use a scintillation counter to measure radioactivity in the medium and in the cell pellet. If the secretary vesicles are being exocytosed, radioactivity should decrease in the medium as leucine is being incorporated into the cell (radioactivity will increase in the pellet) but then should increase in the medium as vesicles are releasing their contents to the cell exterior.

QUESTION #3: Do Mtb-containing vacuoles not fuse w/ lysosomal enzyme-containing vesicles because the vacuoles lack ARF5? (KL/JMC)

1 am going to address the question of whether or not M-tuberculosis-containing vacuoles are able to evade lysosomal fusion because they (the M-tb vacuoles) lack ARF5? The mechanism in which M-tb was internalized is not important. The main issue is that M-tb is able to avoid recognition by ARF5 and subsequently lysosomal fusion. Briefly, ARF is one of eight polypeptides that are known to initiate and compose the protein coat of vesicles which are to be trafficked within the cell. ARF is a GTP-binding protein which is mainly found in the cytosol and co-assembles on the surface of a "donor membrane" to initiate vesicle budding. After the vesicle had budded and is fully coated, ARF is also thought to be involved in the disassembly of the coat and in concert with other membrane proteins, ultimately directing the vesicle to the "acceptor" membrane. There is actually a family of ARF proteins, and ARF5 in particular has not yet been mapped to a distinct part of the cell. For the purpose of this answer, I am assuming that recognition of a donor membrane by ARF5 will subsequently direct that membrane to the acceptor membrane of the lysosome.

The question remains, by what mechanism does M-tb avoid recognition of ARF5? There are several mechanisms in which M-tb could accomplish this, either by secreting a factor that aids in its protection. or M-tb vacuole contains a protein directly on its surface that does not pen-nit recognition by ARF5.

The first step of this experiment would be to establish whether or not ARF5 is even able to associate with the M-tb vacuole. After different time constraints, I would fix my cells (with paraformaldehyde) and add FITC-labeled anti-ARF5 Ab to see if it associates with the M-tb vacuole. If it does bind it, and lysosomal fusion does not occur, then it must be some other protein that is responsible for directing the fusion process. If ARF5 is not found on the M-tb vacuole, it is possible that it mediates lysosomal fusion, but how the M-tb is able to evade ARF5 recognition remains to be seen in the following experiments.

Next I would set up a system using human macrophages for my experiments. My positive control would be to show that M-tb is capable of being internalized and able to sustain life by evading the usual hostile environment of the macrophage. To do this I would first FITC-label M-tb and allow them to become phagocytosed (3 hours, 37 degrees). Next I would quench the cells with Trypan Blue (a vital dye), which would indicate which M-tb were actually inside the cell. By fluorescence microscopy I would see bright green M-tb's, which were inside the mac, and brownish M-tb's that took up the Trypan Blue because weren't internalized and were dead. I would also label the ARF5 G-protein with a rhodamine tag to monitor it's interaction with M-tb. After observing them every day for a week, I will have established that M-tb is capable of surviving what is typically a hostile environment, possibly by the mechanism of evading ARF5 & lysosomal fusion.

Next I would FITC label heat killed M-tb, incubate them with the Macs, and see how they fare. If they are degraded, then it is very possible that the vital M-tb secreted a protein which is integral to it's survival. If the heat-killed organism maintain it's integrity, it is possible that there is a protein in it's membrane that is key to avoiding ARF5.

If in the previous experiment, the heat-killed M-tb did not survive, my next approach would be to attempt to find the protein(s) that are secreted that helped it avoid ARF5. I would look at the M-tb's genome and systematically make point mutations in it's DNA until I mutated the sequence that encoded the key secreted factor that helps it fool ARF5 and ultimately lysosomal fusion. (After each deletion I would incubate that FITC labeled mutant and with the rhodamine labeled ARF5 and monitor their interaction) If I should happen to make a deletion that would cause the ARF5 to recognize and bind the M-tb mutant and subsequently coat and direct it to the lysosome, I would again go back to the M-tb genome, but this time to obtain the sequence that was affected by the mutant and synthesize a functional version of that protein. Next I would incubate the macs with the heat-killed M-tb, but also add my synthesized protein. If I had isolated the appropriate protein, then it would protect the heat killed M-tb from ARF5.

Another possibility would be that the heat-killed M-tb's from my initial experiment were able to avoid recognition by ARF5. This would indicate that the survival mechanism is not a secreted factor, but possibly a protein in the found on the surface of M-tb. I would approach this by first trypsinizing the FITC-labeled M-tb so that they were stripped of all their surface proteins. I would then opsonized them by coating with complement so they could get internalized. The M-tb's inside the mac's are now essentially "bald." Without surface proteins, several possibilities could evolve. Either the M-tb now lacks the surface protein that gave it protection from ARF5 and it will now be recognized, or without it's usual method of entry (recognition by a macrophage receptor), a signal cascade could not be initiated, and therefore the mechanism of survival obliterated. If stripping all the ligands from the surface of the M-tb vacuole led to the recognition by ARF5 (seen by fluorescent microscopy), then the next task would be to try and find which protein is ARF5's ligand. I would do the techniques mentioned above, mutating the proteins that are expressed at M-tb's surface to find which one is bound by ARF5.

Question 4. Can import of cytoplasmic tRNA into mitochondria be driven by a pH gradient? (JC/JMC)

Important Questions:

Hypothesis: Import of tRNA into mitochondria from the cytosol can be driven by a D pH.

Introduction: Much is known about transport of proteins from the cytosol into the mitochondria via transport complexes such as TOM and TIM. Several of the experiments used to determine the factors that are required for protein transporters to work can also be applied to tRNA transport if modified to fit the particularities of RNA.

Materials and Methods:

1 Prepare or purchase radio labeled tRNA

7. View gel using autoradiography.

10. Repeat steps 4 through 7.

Procedure 1: Production and Isolation of radiolabeled met and trp tRNAs. Yeast were pulsed with hot UTPs for 20 min, followed by a chase with excess cold UTPs for 20 min. Cells were harvested and lysed in RNA extraction buffer and digested with Proteinase K, followed by protein removal phenol/chlorofonn extraction, resuspension in DNAse I containing placental RNase inhibitor, EDTA, and SDS. RNA was collected after centrifugation @ 12,000xg for 5 min and redissolved in TE. mRNAs were removed from the total RNA after passing them over an oligo-dT linked Sepharose affinity column. tRNAs were further purified (met and trp specifically) by HPLC using elution over a Porex c-4 column.

In vitro assessment of met and trp tRNA import into mitochondria. Mitochondria (mt) were purified from yeast using first, rate-zonal centrifugation and then density-gradient centrifugation. This was followed by determination of organelle purity using cytochrome c as an indicator of mt fraction purity in conjunction with known enzyme exclusion tests from other organelles. mt were resuspended in an energizing buffer (ATP, pH-cytosolic, of 7-7.5). To the mitochondria were added the following: nothing (control), prepared radiolabeled tRNAs (import test), labeled tRNAs plus RNase A at time points (0, 2, 5, 10, 30, 60 minutes) after tRNA introduction, labeled tRNAs plus trypsin at same time points (check for mt membrane import proteins/receptors for the tRNAs), labeled tRNAs in mitochondria preincubated with valinomycin and K+ in buffer (ionophore that destroys electrochemical gradient), labeled tRNAs in mitochondria preincubated with nigericin (destroys pH gradient), and mitochondria in nonenergizing medium (no ATP).

Assessment and quantitation of import was determined by fractionation and SDS PAGE of mt extracts followed by incubation with the mouse anti-tRNA mabs, and secondary rabbit anti-mouse IgG mabs conjugation and staining in appropriate substrate and subsequent autoradiography and use of analytical software to quantify band intensity.

#4: Can the import of TRNA into mitochondria from the cytosol be driven by a D pH? (VD)

Assay (in vitro): Perform an vitro assay for the import of TRNA using isolated mitochondria, which has been earlier proven to be efficient and reproducible. Suspend freshly isolated mitochondria from culture in a mixture of sorbitol, HEPES, MgCl,, DTT, PMSF, ATP, PEP and PK. After 5 mins of preincubation, mix aliquots with labeled tRNAs and appropriate volume of protein fraction (HEPES, PMSF, DTT and glycerol too). Incubate for appropriate time at 30 deg and then add pancreatic Rnase and phosphodiesterase. The isolated mitochondria is then layered onto sucrose gradient and centrifuged, band recovered and organelles resuspended in buffer to be pelleted, TRNA extracted and electrophoresed (using polyacrylamide gel electrophoresis. The dried gel is exposed to X ray film at -70 deg with intensifying screens)

Assay (in vivo): This can also be performed based on electroporation of intact yeast cells (S. cerevisiae), used to introduce labelled tRNAs into the cytoplasm. (Details in Nuc. Acid Res., 20: 1277-1281, 1992). Determination of D pH

Estimate the magnitude of the prevailing D pH by using 9aminoacridine (9-AA) fluorescence quenching.

The D pH can then be calculated using the fractional quenching, q.

Use the assay technique described above supplying the requisite materials to the incubation mixture and incubate them for different time intervals (2-8 hours). Analyze samples directly after incubation or after incubation of the mitochondria with thermolysin for 30 niins on ice. Estimate D pH by 9-AA fluorescence quenching and use a measuring beam of intensity 0. 1-0.2 watts/m. The D pH is driven by a quartz halogen lamp filtered through a Coming glass filter. Calculate D pH according to the following equation:

D pH = log[q/(I-q)] + log V/v where V is total reaction volume and v is the intemal volume of the mitochondria. The magnitude of D pH is altered by changing the intensity of the incident light, and import reactions are carried out in a random order to obviate any effects due to changes in the import competence of the mitochondria during storage on ice.

Study of rate of import as a function of D pH

Isolate mitochondria and incubate with tRNA (Along with requisite proteins and factors) under varying light intensities (0.05-160 watts/m') Carry out an import assay using nigericin: Remove samples at different time intervals and analyze directly (-protease) or after protease treatment of the mitochondria (+protease). The mature size bands in the fluorograms are to be quantified using laser densitometry. Plot graphs of %added precursor imported v/s time and varying values of D pH. Observe the trend to conrlude if ApH makes contributes to import. Have a control sample which performs the import in the normal manner (Assay procedure listed above - note its rate of import)

Treat mitochondria with carbonyl cyanide m-chlorophenylhydrazone which causes a dissipation of the electrochemical potential via its ability to transport protons across the mitochondrial inner membrane or dissipate the membrane potential using treatment with valinomycin in the presence of KCI or oligomycin in the presence of KCN. Normally, all these 3 treatments would lead to a loss of the capacity of mitochondria to import TRNA in vitro (use this as control). Perform D pH studies on this system and compare results.

IsD pH the driving force for translocation, or does it somehow activate the translocation machinery? Localize tRNA after import into intact mitochondria in the presence of increasing concentrations of nigericin. Carry out parallel time course analyses of the import of tRNA by isolated mitochondria and add nigericin midway through one of the import reactions. IF nigericin rapidly and completely inhibits tRNA transport, we can conclude that the D pH is continuously required for protein translocation to occur. Run similar expts after blocking of the electrochemical potential gradient too. In each case use controls of the original assay without incorporating the newly added step.

NOTE: Use acetate/formate/benzoate ion in case of basic organelles. Unsure about mitochondria:

acidic/basic??

QUESTION 5: Does transportin function as a nuclear export signal (NES) receptor, in addition to functioning as a nuclear localization sequence (NLS) receptor? (KL)

Previous experiments: hnRNP Al shuttles continuously between the nucleus and cytoplasm (Michael, 1995) and M9 = 38 aa domain of Al that localizes Al to nucleus (Pollard, 1996). Transportin binds the M9 domain of A1 and serves as the nuclear transport receptor responsible for the import of A1 into the nucleus (Nakielny et al ‘96).

Al. Does Al bind to transportin? Protein binding assay - Prepare an affinity column containing purified Al protein (Nakielny, 1996) incubated with glutathione, Sepharose and binding buffer. Add purified transportin (Nakielny, 1996) and allow to bind. Elute by boiling in SDS-sample buffer. Analyze eluted fractions using SDS-PAGE and Coomassie staining. For controls, load separate wells with AI only, transportin only, and with both. If Al and transportin interact, should see two bands in the eluted fraction that are the same as the combined control. Can also mutate transportin and perform the same assay. The mutated transportin should decrease or eliminate binding. (As an alternative to better mimic in vivo conditions, pass cellular homogenate over a column containing purified AI protein and follow the above procedure. Include transportin as a control in the gel and determine if binding occurred and if the eluted protein that bound to Al is transporting.

Yeast two hybrid system: protein-protein interactions: Synthesize 1 plasmid (TR-P+) containing Al and a DNA binding domain and another plasmid (LEU+) containing transportin and an activation domain. Cotransfect the plasmids into mutated yeast cells (trp-, leu- and his-) and select on media that is trpand leu-. Only cells containing both plasmids will survive. Then plate cells selected on the trp- and leu- on his- medium colonies form only if Al and transportin interact. Also test for interaction of mutant transportin with Al M9 sequences.

A2. Does transportin mediate nuclear import of hnRNP Al? Nuclear import assay - Permeabilize HeLa cells with digitonin. Incubate M9 sequences and complete Al proteins either with reticulocyte lysate (cytosol source) or with buffer or with purified transportin. Add ATP to all incubations. Fix the cells and immunostain with anti-Al Ab conjugated to fluorescein and examine with a confocal fluorescent microscope. Examine cells immediately after incubation (before import could occur) and after an adequate time for import. If Al is imported, this assay demonstrates that transportin recognized M9 in a native context and that it can import full-length hnRNP Al.

B 1. Is transportin located in the nucleus? Fluorescence microscopy - Permeabilize HeLa cells with digitonin and add anti-transportin Ab tagged with fluorescein and examine with a confocal fluorescent microscope to see if any part of the nucleus is fluorescent. Western blot - Lyse HeLa cells and use differential centrifugation to extract nuclei. Use a detergent to disrupt the nuclear membrane and use SDS-PAGE on the nuclear extract. Transfer to a nitrocellulose membrane, incubate with anti-transportin Ab and autoradiograph.

B2. Do transportin and Al bind in the nucleus? Protein binding assay - Prepare an affinity column containing purified AI protein incubated with glutathione, Sepharose and binding buffer. Add nuclear extract and allow to bind. Elute by boiling in SDS-sample buffer. Analyze eluted fractions usingSDS-PAGE and Coomassie staining. For controls, load separate wells with Al only, transportin only, and with both. If the two proteins interact in the nucleus, should see two bands in the eluted fraction that are the same as the lane containing the combined control.

B3. Does transportin mediate nuclear export and import? Nuclear export assay = Transient transfection interspecies heterokaryon assay system (Michael, 1995). Transfect HeLa cells with an epitope tagged expression construct to induce the expression of a protein and then fuse to mouse NIH 3T3 cells to form heterokaryons. After I hr, fix and stain for immunofluorescence microscopy. If migration occurred out of the heterologous nuclei, should detect a signal within the mouse nucleus of the heterokaryon. Hoechst staining is used to differentiate between mouse and human nuclei.

Transfect with the following Myc tagged constructs into HeLa cells known to contain transportin (Northern blot):

AI (with intact M9 sequence)

pyruvate kinase (cytoplasmic protein) PK + M9 PK + SV40 large T Ag NLS

nucleoplasmin core domain (restricted to nucleus) NCD + M9 NCD + SV40 large T Ag NLS

Repeat with over-expressed mutated transportin (dominant negative). If these constructs are transported in and out of the nucleus with endogenous transportin but no longer migrate if transportin is mutated, then transportin is a NLS and NES receptor. (Alternatively, use the same constructs but use microinjection into the nucleus and cytoplasm of Xenopus laevis oocytes).

Question 5: Does transportin function as a nuclear export signal (NES) receptor, in addition to functioning as a nuclear localization sequence (NLS) receptor? (MC-S)

It is known that transportin functions as a NLS receptor for hnRNP Al, binding to the 38 amino acid sequence known as M9 and is "necessary and sufficient" for temperature- and energy-dependent nuclear import. It has also been shown that this same aa sequence mediates both import and export. Studies designed to delineate these import and export functions by mutagenesis of highly conserved residues have been unsuccessful, with point mutations blocking either both or neither import/export. It is a reasonable assumption that the import and export signals are most likely the same within M9, and if so, that its receptor, transportin, might have a role in export as well as import. The following experiments are

designed to test this hypothesis.

Microinjection into Xenopus oocytes of small immunogold-labeled recombinant protein (such as the PK artificial protein or a protein normally confined to the nucleus, nucleoplasmin) including the M9 sequence, along with large immunogold-labeled transportin. (Assuming preparation and controls for the antibodies conjugated to colloidal gold) Using immunoelectron microscopy, examine colocalization as well as quantification to determine ratio of large to small gold. To allow for different shuttling rates between M9 and transportin, quantification could be performed at different time points to see if the ratio changes. To see if transportin is required, the large immuno-gold label could be attached to nonfunctional transportin (binds M9, but can't leave nucleus).

Another system that could complement the more expensive and cumbersome microinjection procedure, is the use of a "transient transfection interspecies heterokaryon assay system". HeLa cells are transfected with epitope-tagged expression constructs for the proteins of interest (Al-M9, transportin) and are fused with mouse N-IH 3T3 cells (in the presence of cyclohexamide) to form heterokaryons. One hour postfusion, the cells can be fixed and stained for immunofluorescence microscopy to determine the distributionoftaggedAl-M9andtransportin. Iftheybothendupinthemousenucleusofthe heterokaryon, it demonstrates that both were exported and re-imported into the new nucleus. If performed in the absence of a functional transportin receptor protein and Al -M9 can still get out of the nucleus, but not into the mouse nucleus, it may be an indication of a separate NES receptor, other than or in addition to, transportin. (Would need to deal with the issue of endogenous transportin)

In order to see what other sorts of nuclear proteins bind to M9, it would also be interesting to use M9 affinity chromatography with nuclear extracts and gel electrophoresis to separate and subsequently purify proteins in the bound fraction. It would provide information on how specific M9 binding is, and any successfully isolated/purified M9-binding proteins could be used in the assays above for their role in export. (Yeast two hybrid systems were used to identify transportin and provide another technique for identifying important export mediating proteins).

#5: Does transportin function as a nuclear export signal (NES) receptor, in addition to functioning as a

nuclear localization sequence (NLS) receptor? (VD)

To prove ability of transportin (TPT) to bind to leucine-rich sequence (NES) proteins (e.g. Rev protein)

Expt 1: Mix ³⁵S Methionine and ³⁵S cysteine labeled human MYC-tagged transportin translated in vitro with ³⁵S Methionine and ³⁵S cysteine labeled SV5 tagged wild type (wt) and mutated (mut) NES protein (e.g. Rev protein)(with leucine sequences replaced by alanine) translated in vitro. Process for immunoprecipitation with anti-MYC tag or an anti-SV5 tag. Analyze inununoprecipitates by using 7% SDS-PAGE and autoradiography.

Co-precipitation of transportin and wt protein with both antibodies and not when mut protein is used is indicative of interaction.

Expt 2: Incubate 15S Methionine and "S cysteine labeled transportin translated in vitro with streptavidin-agarose beads bound to biotinylated bovine serum albumin-NES (biot-BSA-NES) or mut NES peptides. Collect the bound and unbound fractions and analyze by 7% SDS-PAGE and autoradiography.

Observe for binding on autoradiograph. Try replacing leucine residues of NES with alanines or replacing NES with NLS sequence, lack of binding on autoradiograph would indicate interaction.

Expt 3: This expt would be performed if an inhibitor of transportin is obtained, which would then compete with the NES protein (e.g. Rev protein) for transportin. Incubate "S Methionine and "S cysteine labeled transportin with streptavidin-agarose beads bound to biotinylated bovine serum albumin-NES conjugate (30 min at room temperature in PBS) while increasing the concentration of the inhibitor. Collect the bound and unbound fractions and analyze using 7% SDS-PAGE and autoradiography. Blockage of formation of transportin-NES complex is indicative of interaction.

Assay: HeLa cells are transfected with cDNAs encoding fusion proteins consisting of MYC-tagged pyruvate kinase (PDO, wt or mut NES protein (e.g. Rev protein) and SV40 large T antigen NLS to direct the resulting proteins to the nucleus (NLS-PK-NES and NLS-PK-NESmut resp.). Eighteen hours after transfection, the cells are permeabilized using digitonin in transport buffer and incubated for 30 min at 23'C with BSA in transport buffer in the absence (buffer) or presence of ATP (buffer + ATP) or with X. laevis egg extracts (cytosol) in transport buffer in the presence of ATP (extracts + ATP) or in the absence of ATP (addition of apyrase; extracts + apyrase). Incubate and process cells for immunofluorescence or for protein immunoblotting. In both cases detect NLS-PK-NES and NLS-PK-NESmut with monoclonal anti-MYC tag. Visualize nuclear DNA using DAPI stain. Use hnRNP as an internal control (non-exported protein) in the same samples.

In vitro nuclear export of NLS-PK-NES performed as above, using X. laevis egg extracts supplemented with ATP(extracts + ATP), or in extracts supplemented with ATP and the inhibitor (extracts + ATP + inhibitor). Examine nuclear export of NLS-PK-NES using indirect immunofluorescence or protein immunoblot using an anti-MYC tag or an anti-hnRNP C.

This method is just explained in brief. One could introduce a NES-GFP-NLS reporter into a TPT deletion strain of S. cerevisiae expressing either the wt or the mut TPT grown either at RT or shifted to 37 deg for 15 mins. To test the specificity of the export phenotype in mut TPT cells, the localization of the protein exporter reporter can be examined in RIP 1 deletion strain and wt strain.(RIP I binds to NES in a two hybrid assay). Based upon the results we could conclude if nuclear export is impaired in mut TPT cells, which would indicate their role as NES receptor.

Point mutations in the M9 sequence of hnRNPA abolish both the import and export of the protein, indicating that M9 is involved in both processes. If M9 is involved in export, does transportin mediate this step too? A very simple experiment was carried out using yeast protein, wherein a temperature sensitive mutant of Kapl04p (functional analog of transportin) was isolated. At the non-permissive temperature, levels of Kapl04p declined rapidly in the cell and the caused re-distribution of exported protein in the cytoplasm, however since only a weak reduction of this protein was observed (Nab2p), it was concluded that Kap 104p is primarily involved in nuclear import and not export. Note: The similarities of CRM 1 (NES receptor) to transportin create a distinct possibility that this too is a NES receptor.