She3p Possesses a Novel Activity Required for ASH1 mRNA … · ASH1 mRNA is proposed to be statically anchored through a mechanism requiring translation of ASH1 mRNA (5, 26, 33)

EUKARYOTIC CELL, July 2009, p. 1072–1083 Vol. 8, No. 71535-9778/09/$08.00�0 doi:10.1128/EC.00084-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

She3p Possesses a Novel Activity Required for ASH1 mRNALocalization in Saccharomyces cerevisiae�

Sharon M. Landers, Michelle R. Gallas, Jaime Little, and Roy M. Long*Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee,

Wisconsin 53226

Received 12 March 2009/Accepted 1 May 2009

Intracellular and intercellular polarity requires that specific proteins be sorted to discreet locations withinand between cells. One mechanism for sorting proteins is through RNA localization. In Saccharomyces cerevi-siae, ASH1 mRNA localizes to the distal tip of the bud, resulting in the asymmetric sorting of the transcrip-tional repressor Ash1p. ASH1 mRNA localization requires four cis-acting localization elements and thetrans-acting factors Myo4p, She3p, and She2p. Myo4p is a type V myosin motor that functions to directlytransport ASH1 mRNA to the bud. She2p is an RNA-binding protein that directly interacts with the ASH1mRNA cis-acting elements. Currently, the role for She3p in ASH1 mRNA localization is as an adaptor protein,since it can simultaneously associate with Myo4p and She2p. Here, we present data for two novel mutants ofShe3p, S348E and the double mutant S343E S361E, that are defective for ASH1 mRNA localization, and yetboth of these mutants retain the ability to associate with Myo4p and She2p. These observations suggest thatShe3p possesses a novel activity required for ASH1 mRNA localization, and our data imply that this functionis related to the ability of She3p to associate with ASH1 mRNA. Interestingly, we determined that She3p isphosphorylated, and global mass spectrometry approaches have determined that Ser 343, 348, and 361 are sitesof phosphorylation, suggesting that the novel function for She3p could be negatively regulated by phosphory-lation. The present study reveals that the current accepted model for ASH1 mRNA localization does not fullyaccount for the function of She3p in ASH1 mRNA localization.

RNA localization is a process to spatially and temporallyrestrict intracellular and intercellular protein expression. Thismechanism for protein sorting is utilized by a number of eu-karyotic organisms including mammals, Drosophila melano-gaster, Xenopus spp., and Saccharomyces cerevisiae (4, 15, 25,37, 44). RNA localization can be achieved through at leastthree distinct mechanisms: cytoplasmic diffusion followed byentrapment of the RNA at the site of localization, generalizeddegradation/localized protection, and directed transport usingmotor proteins and cytoskeletal elements (60). S. cerevisiaeutilizes directed transport to localize at least 30 mRNAs to thebud tip (2, 3, 57, 61). Localization of ASH1 mRNA to the budtip in yeast results in the asymmetric sorting of Ash1p to thedaughter cell nucleus (10, 43, 62). Ash1p is a transcriptionalrepressor that prevents expression of the HO endonuclease inthe daughter cell, restricting mating-type switching exclusivelyto the mother cell (6, 36, 45, 58).

ASH1 mRNA localization is dependent on She2p, She3p,and Myo4p (43, 62). She2p is an RNA-binding protein thatassociates with the four ASH1 mRNA cis-acting localizationelements: E1, E2A, E2B, and E3 (7, 11, 26, 41, 48). She2p alsodirectly associates with the C terminus of She3p (7, 11, 26, 41,48). The N terminus of She3p has the ability to associate withthe type V myosin, Myo4p (7, 19, 47). Furthermore, She3p cansimultaneously associate with Myo4p and the She2p-ASH1mRNA complex (21, 24). These observations led to the current

model for ASH1 mRNA localization in which the simultaneousassociation of She3p with Myo4p and the She2p-ASH1 mRNAcomplex results in the formation of a transport particle thatmoves along the polarized actin cytoskeleton into the daughtercell (7, 41, 63). Once the transport particle reaches the bud tip,ASH1 mRNA is proposed to be statically anchored through amechanism requiring translation of ASH1 mRNA (5, 26, 33).

Superimposed on the mRNA localization machinery areproteins required for repressing translation of ASH1 mRNAuntil the mRNA reaches the site of localization. Khd1p andPuf6p are RNA-binding proteins that function to repress trans-lation of ASH1 mRNA, and the RNA-binding activities forKhd1p and Puf6p are negatively regulated by phosphorylation(16, 28, 33, 50). In addition to its function in ASH1 mRNAlocalization, She2p also has a role in repressing translation ofASH1 mRNA. She2p associates with ASH1 mRNA in the nu-cleus, and the nuclear association of She2p with ASH1 mRNAhas a role in ASH1 mRNA localization and translational re-pression of ASH1 mRNA by recruiting Loc1p and Puf6p toASH1 mRNA in the nucleus (18, 56).

Given the importance of phosphorylation in regulating theactivities of Khd1p and Puf6p, we investigated whether phos-phorylation of She3p has a role in the mechanism of ASH1mRNA localization. We determined that She3p is phosphory-lated in vivo, and we observed that She3p phosphorylationmutants S348E and the double mutant S343E S361E retain theability to associate with Myo4p and She2p but are unable tolocalize ASH1 mRNA. Given that the only known functions forShe3p in ASH1 mRNA localization are the ability to associatewith Myo4p and She2p, our results suggest that phosphoryla-tion of She3p negatively regulates a novel ASH1 mRNA local-ization activity.

* Corresponding author. Mailing address: Department of Microbi-ology and Molecular Genetics, Medical College of Wisconsin, 8701Watertown Plank Rd., Milwaukee, WI 53226. Phone: (414) 456-8423.Fax: (414) 456-6535. E-mail: [email protected].

� Published ahead of print on 8 May 2009.

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MATERIALS AND METHODS

Yeast strains, media, and plasmids. Yeast cells were grown in defined syn-thetic media lacking the indicated nutrients or rich YEP medium (53). All mediacontained 2% glucose unless indicated otherwise. Yeast cells were transformedby using lithium acetate (34). As described elsewhere, yeast strains deleted ofnonessential genes were created by using PCR products generated from plasmidpUG6 (30). To label yeast cells with [32P]orthophosphate, minimal D3/5 high-Pi

medium lacking leucine and SMD low-Pi medium were used (42). The yeaststrains used in the present study are listed in Table 1.

Plasmids used in the present study are listed in Table 2 and were created byusing standard techniques (54). SHE3 mutants were generated by overlap PCR(13). Briefly, four oligonucleotide primers were used per mutant in two sequen-tial rounds of PCR. During the first round of PCR, two overlapping PCRproducts were amplified, and both contained the desired mutation in the regionof overlap between the two PCR products. The two PCR products were gelpurified using the Promega Wizard SV Gel and PCR clean-up system. Thetemplate for the second round of PCR consisted of the two initial PCR productsbeing combined in equivalent concentrations. All mutations within SHE3 wereconfirmed by DNA sequencing. Further information regarding the constructionof specific plasmids is available upon request.

In vivo labeling and immunoprecipitation. In vivo labeling of yeast cells with[32P]orthophosphate was performed as described previously (42). Briefly, yeastcells were grown overnight at 30°C in minimal D3/5 high-Pi medium lackingleucine to an optical density at 600 nm (OD600) of 0.600. Subsequently, thecultures were diluted into fresh SMD low-Pi medium to an OD600 of 0.4. Yeastcells were grown for 4 h at 30°C in SMD low-Pi medium containing 0.5 mCi of[32P]orthophosphate/ml. After labeling, the cells were harvested by centrifuga-tion, cell lysates were prepared by breaking the cells with glass beads in 500 �l ofbreaking buffer (100 mM NaHPO4 [pH 7.4], 1 M NaCl, 10% Triton X-100, 1%sodium dodecyl sulfate [SDS], 5% deoxycholate, 0.1 �g of chymostatin/ml, 2 �gof aprotinin/ml, 1 �g of pepstatin/ml, 0.5 �g of leupeptin/ml, 0.01 �g of benza-midine/ml, 1 mM Na3VO4, 50 mM Na3F, 8.67 mg of �-glycerophosphate/ml),and the lysate was recovered from the glass beads. The glass beads were washedwith 200 �l of fresh breaking buffer and combined appropriately. The lysateswere cleared by centrifugation at 16,000 � g for 10 min at 4°C. The solublefraction was recovered and stored at �80°C. For immunoprecipitation, the celllysates were diluted into fresh breaking buffer for a total volume of 1 ml, and 5�l of rabbit polyclonal She3p antiserum was added. She3p immune complexeswere allowed to form by incubation at 4°C for 2 h with rotation. Subsequently,She3p immune complexes were collected with protein A-agarose beads. The

TABLE 1. Yeast strains used in this study

Strain Genotype Source orreference

K699 MATa ade2-1 his3-11 leu2-3,112 ura3 trp1-1ho can1-100

36

PJ69-4a MATa his3-200 leu2-3,112 trp1-90 ura3-52gal4 gal80 GAL2-ADE2LYS2::GAL1-HIS3 met2::GAL7-lacZ

35

YLM584 MATa ade2 his3-200 leu2-3,112 trp1-1 ura3-52 LYS2::(LexAop)-lacZ LexA-MS2-MS2coat (N55K) she1::KAN

41

YLM677 MATa ade2-1 his3-11 leu2-3,112 ura3 trp1-1ho can1-100 she3::KAN

This study

YLM923 MAT� ade2-1 leu2-3 trp1-1 ura3 HO-ADE2HO-CAN1 she3::KAN

This study

YLM1320 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3ho can1-100 she2� silent she3A silentMyo4p-3HA::kanMX6

23

YLM1406 MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3ho can1-100 she2� silent she3A silentMyo4p-myc13::kanMX6

23

TABLE 2. Plasmids used in this study

Plasmid Features Source or reference

pACT2 Yeast vector for expressing Gal4p activation domain fusion proteins 39pET21b E. coli vector for generating His6-tagged fusion proteins NovagenpGBDU-c2 Yeast vector for expression of Gal4p DNA-binding domain fusion proteins 35pUG6 Plasmid for generating KAN disruption cassettes by PCR 30YCplac22 Yeast single-copy shuttle plasmid marked with TRP1 22YCplac111 Yeast single-copy shuttle plasmid marked with LEU2 22YCplac33 Yeast single-copy shuttle plasmid marked with URA3 22YEplac181 Yeast multicopy shuttle plasmid marked with LEU2 22YEplac195 Yeast multicopy shuttle plasmid marked with URA3 22C3431 YEplac195 containing ASH1 43pIIIA/MS2-2 Three-hybrid vector for expressing MS2-fusion RNAs 68pEP13 pACT2 expressing wild-type She3p 41pRL80 pIIIA/MS2-2 containing ASH1 E3 cis-acting localization element 41pRL128 YEplac181 expressing wild-type She3p 41pRL174 pGBDU-c2 expressing wild-type She2p 41pRL189 YEplac181 expressing She3p-myc6 This studypRL200 YCplac111 expressing She3p-myc6 24pRL461 pET21b expressing She3p amino acids 236 to 425 This studypRL519 YCplac111 expressing wild-type She3p This studypRL676 YCplac22 expressing She2p-myc6 23pRL1138 YCplac111 expressing She3p-S343A S361A-myc6 This studypRL1139 YCplac111 expressing She3p-S343E S361E-myc6 This studypRL1143 YCplac33 expressing wild-type She2p This studypRL1182 YCplac111 expressing She3p-S343A S361A This studypRL1183 YCplac111 expressing She3p-S343E S361E This studypRL1206 YCplac111 expressing She3p-S348A-myc6 This studypRL1207 YCplac111 expressing She3p-S348E-myc6 This studypRL1221 pACT2 expressing She3p-S343A S361A This studypRL1222 pACT2 expressing She3p-S343E S361E This studypRL1225 pACT2 expressing She3p-S348A This studypRL1226 pACT2 expressing She3p-S348E This studypRL1252 YCplac111 expressing She3p-S348A This studypRL1253 YCplac111 expressing She3p-S348E This study

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beads were washed three times with 1 ml of fresh breaking buffer/wash. Boundproteins were eluted by boiling the protein A-antibody complexes in Laemmlibuffer. The immunoprecipitations were analyzed by SDS-polyacrylamide gelelectrophoresis (PAGE) and autoradiography.

SHE genetic selection. Plasmids expressing wild-type and mutant versions ofShe3p were transformed into yeast strain YLM923, and transformants wereselected on synthetic medium lacking leucine. Subsequently, single yeast colonieswere picked and grown overnight at 30°C in liquid synthetic medium lackingleucine. Then, 2 �l of the undiluted overnight culture and 10�1, 10�2, 10�3,10�4, and 10�5 dilutions were spotted onto synthetic medium devoid of leucinein the absence and presence of 0.03% canavanine. The plates were incubated at30°C for 2 days, and images were captured. Subsequently, to ensure that the yeastcells produce the various She3p mutant proteins, cell lysates were prepared andanalyzed by Western blotting with an anti-myc 9E10 monoclonal antibody(Roche), as well as with anti-Pgk1p (Molecular Probes).

In situ hybridization. Yeast cultures were grown in appropriate media to earlylog phase, fixed with formaldehyde, and converted to spheroplasts as describedpreviously (9, 43). Subsequently, spheroplasts were hybridized with a pool ofCy3-conjugated ASH1 oligonucleotide probes and stained with DAPI (4�,6�-diamidino-2-phenylindole) (9, 43). Coverslips were mounted on slides with phen-ylenediamine mounting medium (9). Cells were visualized, and images werecaptured on a Nikon Eclipse 600 epifluorescence microscope equipped with a60� N.A. 1.4 objective lens, connected to a Micromax-Interline transfer charge-coupled device camera (Princeton Instruments, Inc.), and using MetaMorphimaging software (Universal Imaging Corp.).

Coimmunoprecipitation assays. Exponentially growing cultures correspondingto 3 � 108 yeast cells were harvested, and soluble lysates were prepared inbreaking buffer containing 50 mM HEPES-KOH (pH 7.3), 20 mM potassiumacetate, 2 mM EDTA, 0.1% Triton X-100, 5% glycerol, 0.1 �g of chymostatin/ml,2 �g of aprotinin/ml, 1 �g of pepstatin/ml, 0.5 �g of leupeptin/ml, and 0.01 �g ofbenzamidine/ml as described previously (23). Protein complexes were purified bythe addition of 7.5 �g of anti-myc 9E10 monoclonal antibody prebound toprotein A-agarose beads (Pierce) and incubated for 2 h at 4°C. The immunecomplexes were collected by centrifugation at 500 � g for 4 min at 4°C. Thematrix was washed four times with 500 �l of wash buffer (50 mM HEPES-KOH[pH 7.3], 20 mM potassium acetate, 2 mM EDTA, 0.1% Triton X-100, 5%glycerol). Bound proteins were eluted by boiling in Laemmli buffer. Equivalentamounts of cell extracts (input) and precipitated sample were separated bySDS-PAGE and analyzed by Western blotting. Proteins containing the hemag-glutinin (HA) epitope were detected with anti-HA11 monoclonal antibody (Co-vance), whereas She2p and She3p were detected with rabbit polyclonal anti-She2p and anti-She3p antiserum, respectively.

Preparation of rabbit polyclonal She3p antiserum. Polyclonal rabbit anti-serum was raised against She3p amino acids 236-425-His6 expressed and purifiedfrom Escherichia coli. Plasmid pRL461 was transformed into E. coli strainBL21(DE3)/pLysS, and expression of She3p-His6 was induced with 1 mM IPTG(isopropyl-�-D-thiogalactopyranoside). Subsequently, the cells were harvested bycentrifugation and resuspended in IMAC-5 (20 mM Tris-HCl [pH 7.9], 500 mMNaCl, 10% glycerol, 0.1% NP-40, 5 mM imidazole) containing 175 �g of phen-ylmethylsulfonyl fluoride/ml, 2 �g of aprotinin/ml, 2 �g of leupeptin/ml, 1 �g ofpepstatin/ml, 100 �g of RNase A/ml, and 10 �g of DNase I/ml. A lysate wasprepared by passing the cell suspension three times through a French press, andinsoluble material was removed from the lysate by centrifugation at 30,000 � gfor 20 min at 4°C. She3p-His6 was purified from the lysate by using Ni-NTA(Qiagen) affinity chromatography and SDS-PAGE. The region of the SDS-PAGE gel corresponding to She3p-His6 was excised, combined with Freundadjuvant, and injected into a female New Zealand White rabbit.

Two- and three-hybrid analyses. The yeast strains PJ69-4a (two-hybrid assay)and YLM584 (three-hybrid assay) were transformed with the indicated plasmids,and transformants were grown in synthetic medium lacking leucine and uracil(35). Quantitative liquid �-galactosidase assays were performed using ONPG(o-nitrophenyl-�-D-galactopyranoside) as previously described (29). Briefly, theyeast cells were grown to mid-log phase in synthetic medium lacking leucine anduracil, and cells were harvested by centrifugation. Subsequently, the cells wereresuspended in 5 ml of buffer Z (60 mM Na2PO4, 40 mM NaHPO4, 10 mM KCl,1 mM MgSO4, and 50 mM �-mercaptoethanol at pH 7.0), and the OD600 wasdetermined in duplicate. An aliquot of the cell suspension was analyzed intriplicate for �-galactosidase activity in 60 mM Na2PO4, 40 mM NaHPO4, 10 mMKCl, 1 mM MgSO4, 50 mM �-mercaptoethanol, 0.0025% SDS, and 5% chloro-form by vortexing the cell suspension for 10 to 15 s, followed by equilibration at30°C for 15 min and incubation with 0.67 mg of ONPG/ml at 30°C for 30 min.The reactions were terminated by the addition of 300 mM Na2CO3 and centri-fuged for 5 min at 1,100 � g. Subsequently, the OD420 and OD550 of the

supernatants were determined, and the �-galactosidase activity was calculated byusing the following formula: U 1,000 � [OD420 � (1.75 � OD550)]/[time (inmin) � volume of the aliquot of the cell suspension (in ml) � OD600]. The�-galactosidase values reported represent the average of three independentexperiments.

Immunoprecipitation–reverse transcription-PCR (IP/RT-PCR) analysis. Im-munoprecipitations of She3p-myc6 and detection of the associated mRNAs wereperformed essentially as described previously (23, 33). In summary, 3 � 108

exponentially growing yeast cells were harvested by centrifugation and disruptedwith glass beads in 400 �l of breaking buffer containing 25 mM HEPES-KOH(pH 7.5), 150 mM KCl, 2 mM MgCl2, 200 U of Superase-In (Ambion)/ml, 0.1%NP-40, 1 mM dithiothreitol, 0.2 mg of heparin/ml, 0.1 �g of chymostatin/ml, 2 �gof aprotinin/ml, 1 �g of pepstatin/ml, 0.5 �g of leupeptin/ml, and 0.01 �g ofbenzamidine/ml. Cell lysates were cleared by centrifugation at 4,000 � g for 5min, and the immunoprecipitation was performed at 4°C for 2 h with anti-mycmonoclonal antibody 9E10 prebound to protein A-agarose beads. Immune com-plexes were recovered by centrifugation and washed four times in 500 �l of washbuffer (25 mM HEPES-KOH [pH 7.5], 150 mM KCl, 2 mM MgCl2). Protein-RNA complexes were eluted from protein A-agarose beads at 65°C for 10 min in100 �l of elution buffer (50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 10 mMEDTA, 1% SDS). An aliquot of the elution was saved for Western blotting. RNAwas extracted from the remaining portion of the sample by using phenol-chlo-roform–isoamyl alcohol and ethanol precipitation. The resulting RNA pellet wasresuspended in nuclease-free water and treated with DNase. The DNase-treatedRNA was subsequently analyzed with an Access/RT-PCR kit (Promega) usingprimers specific for ASH1 mRNA.

RESULTS

Identification of novel She3p mutants that are defective forASH1 mRNA localization. She3p contains numerous consensusphosphorylation sites, implying that phosphorylation of She3pcould regulate ASH1 mRNA localization activities. We inves-tigated the in vivo phosphorylation state of She3p by labelingcells with [32P]orthophosphate and immunoprecipitatingShe3p (Fig. 1). We observed a 32P-labeled protein correspond-ing to the molecular weight of She3p from an immunoprecipi-tation performed with a wild-type lysate, and this 32P-labeledprotein is absent from an immunoprecipitation performed witha she3� lysate. These results suggest that either She3p is phos-

FIG. 1. She3p is phosphorylated in vivo. (A) Yeast strain K699 wastransformed with plasmid YEplac181 (vector) (wt), while yeast strainYLM677 was transformed with plasmids YEplac181 (she3�), pRL128(YEplac181-She3p) (multicopy She3p), or pRL189 (YEplac181-She3p-myc6) (multicopy She3p-myc6). Transformants were grown in syntheticmedium lacking leucine and metabolically labeled with 32PO4. Lysateswere prepared, and She3p was immunoprecipitated with rabbit poly-clonal She3p antiserum. Subsequently, the immunoprecipitate was an-alyzed by SDS-PAGE and autoradiography. The positions of the mo-lecular mass markers are indicated to the left.

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phorylated in vivo or a protein with a molecular weight similarto that of She3p is phosphorylated in a She3p-dependent man-ner. We further investigated whether She3p is phosphorylatedin vivo by transforming a she3� strain with either a multi-copy plasmid expressing She3p or a multicopy plasmid ex-pressing She3p-myc6 and labeling the transformants with[32P]orthophosphate. After immunoprecipitation with the ex-tract containing She3p (multicopy She3p), we observed a 32P-labeled protein migrating at the predicted molecular weight forShe3p. Furthermore, after immunoprecipitation with theShe3p-myc6 extract (multicopy She3p-myc6), we observed aslower-migrating 32P-labeled protein, corresponding to She3p-myc6, and we observed an absence of a 32P-labeled protein atthe molecular weight corresponding to She3p. From these re-sults we conclude that She3p is phosphorylated in vivo.

To begin to map the sites of phosphorylation within She3p,we performed phosphoamino acid analysis and determinedthat She3p is phosphorylated in vivo predominantly on Serresidues (data not shown). Consistent with our in vivo labelingstudies, global mass spectrometry approaches determined thatSer 28, 217, 343, 348, 361, 392, and 394 are sites of phosphory-lation within She3p (1, 12, 27, 40, 59). In addition, Thr 393 andTyr 123 have been reported to be sites of phosphorylationwithin She3p (1, 51). Ser 28 and Tyr 123 map to the region ofShe3p that interacts with Myo4p (Fig. 2A) (7). Ser 217 maps toa region between the domains of She3p that associate witheither Myo4p or She2p, while Ser 343, 348, 361, 392, 394, andThr 393 map to the region of She3p that interacts with She2pand ASH1 mRNA in a She2p-dependent manner (Fig. 2A)(7, 41).

Using the SHE genetic selection, we investigated whetherSer phosphorylation has a regulatory role in ASH1 mRNAlocalization by individually converting the Ser residues to Ala,which prevents phosphorylation, or Glu, which mimics consti-tutive phosphorylation. In the SHE genetic selection the asym-metric regulation of the HO promoter is monitored by thereporter gene HO-CAN1 (6, 36). Consequently, in the SHEgenetic selection yeast colonies with a defect in ASH1 mRNAlocalization have the ability to grow in the presence of the toxicarginine analog canavanine. From our analysis, we observedthat the She3p-S348E mutant is the only single Ser amino acidsubstitution that is insensitive to canavanine (Fig. 2B and Table3 and data not shown). We also investigated whether multipleSer-to-Ala and Ser-to-Glu substitutions alter the ability ofShe3p to localize ASH1 mRNA. We observed that the She3p-S343E S361E mutant also is insensitive to canavanine (Fig. 2B,Table 3, and data not shown). These results suggest thatShe3p-S348E and S343E S361E are defective for ASH1 mRNAlocalization activity. In contrast, we observed in the SHE ge-netic selection that every individual Ser-to-Ala substitution ormultiple Ser-to-Ala substitutions exhibited a wild-type phe-notype (Fig. 2B and Table 3).

The SHE genetic selection is an indirect assay to monitor thedistribution of ASH1 mRNA and Ash1p. Therefore, to directlyvisualize the distribution of ASH1 mRNA, we performed flu-orescent in situ hybridization (FISH) for ASH1 mRNA in cellsexpressing wild-type She3p, She3p-S348A, She3p-S348E,She3p-S343A S361A, or She3p-S343E S361E (Fig. 2C). Wecounted anaphase cells with signal for ASH1 mRNA and ob-served that cells expressing She3p-S348A or She3p-S343A

S361A localize ASH1 mRNA as well as wild-type She3p. Incontrast, we observed that cells expressing She3p-S348E orShe3p-S343E S361E are unable to localize ASH1 mRNA asefficiently as wild-type She3p. Furthermore, we observed thatcells expressing She3p-S348E or She3p-S343E S361E have aslight increase in the number of cells exhibiting the full budphenotype which has been attributed to defects in anchoringASH1 mRNA at the site of localization (Fig. 2C) (26, 33). Thesum of our results from the SHE genetic selection and FISHindicate that She3p-S348E and She3p-S343E S361E cells aredefective for ASH1 mRNA localization and Ash1p sorting.

A dramatic increase or decrease in the expression of She3p-S348E and She3p-S343E S361E would provide a trivial expla-nation for the inability of these She3p mutants to localizeASH1 mRNA. Consequently, we investigated the expression ofthe She3p mutants by Western blotting and determined thatShe3p-S348E and She3p-S343E S361E are expressed at equiv-alent levels to wild-type She3p, She3p-S348A, and She3p-S343A S361A (Fig. 3A and data not shown). The sum of ourresults is consistent with the hypothesis that phosphoryla-tion of She3p at Ser 343, 348, and 361 negatively regulatesan ASH1 mRNA localization activity. Furthermore, since weobserved that the corresponding She3p-S348A and She3p-S343A S361A mutants retain the ability to localize ASH1mRNA (Fig. 2B and C and Table 3), we hypothesize thatphosphorylation at these Ser residues is not required forASH1 mRNA localization.

She3p-S348E and She3p-S343E S361E retain the ability toassociate with Myo4p and She2p. She3p possesses two knownactivities required for ASH1 mRNA localization: the ability tointeract with Myo4p and the ability to interact with She2p (7,41, 63). Consequently, we investigated the ability of She3p-S348E and She3p-S343E S361E to associate with Myo4p andShe2p by coimmunoprecipitation (Fig. 3A). We observed thatwild-type She3p (lane 3), She3p-S348A-myc6 (lane 4), She3p-S348E-myc6 (lane 5), She3p-S343A S361A-myc6 (lane 6), andShe3p-S343E S361E-myc6 (lane 7) immunoprecipitate equiva-lent amounts of Myo4p-3HA and She2p. We further studiedthe She3p-She2p interaction by two-hybrid analysis. She3p-S348A, She3p-S348E, She3p-S343A S361A, and She3p-S343ES361E express She2p-dependent LacZ activity at levels com-parable to that of wild-type She3p (Fig. 3B). Taken together,these results suggest that the observed ASH1 mRNA localiza-tion defect for She3p-S348E and She3p-S343E S361E is notdue to defects in the Myo4p or She2p interactions.

In the proposed model for ASH1 mRNA localization, She3psimultaneously associates with Myo4p and She2p. WhileShe3p-S348E and She3p-S343E S361E mutants retain the abil-ity to individually associate with Myo4p and She2p, it remaineda formal possibility that these She3p mutants may not retainthe ability to simultaneously associate with Myo4p and She2p.Consequently, we investigated whether She3p-S348E andShe3p-S343E S361E could simultaneously associate withMyo4p and She2p by coimmunoprecipitation (Fig. 4). We ob-served that each of the She3p mutants and She2p coimmuno-precipitate with Myo4p-myc13. These results reinforce our as-sertion that the ASH1 mRNA localization defect in yeaststrains expressing She3p-S348E or She3p-S343E S361E doesnot result from the inability of these She3p mutants to associ-ate with Myo4p or She2p. Furthermore, these results imply



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that She3p contains an unidentified activity required for ASH1mRNA localization.

She3p-S348E and She3p-S343E S361E are defective for as-sociation with ASH1 mRNA. The current model for ASH1

mRNA localization postulates that She3p is tethered to ASH1mRNA through a protein-protein interaction with She2p (7,41, 63). Since She3p-S348E and She3p-S343E S361E retain theability to associate with She2p, we reasoned that these She3p

FIG. 2. ASH1 mRNA is delocalized in cells expressing She3p-S348E or She3p-S343E S361E. (A) Domain diagram for She3p. Amino acids 1to 197 of She3p contain a domain required for the association with Myo4p, while amino acids 231 to 425 of She3p contain a domain required forthe association with She2p and ASH1 mRNA. Sites of phosphorylation are also indicated. (B) Yeast strain YLM923 was transformed with plasmidsYCplac111 (vector) (row 1), pRL200 (She3p-wt-myc6) (row 2), pRL1206 (She3p-S348A-myc6) (row 3), pRL1207 (She3p-S348E-myc6) (row 4),pRL1138 (She3p-S343A S361A-myc6) (row 5), or pRL1139 (She3p-S343E S361E-myc6) (row 6). Transformants were spotted on synthetic mediumdevoid of leucine and arginine in the absence (�) or presence (�) of canavanine. (C) Strain YLM923 was transformed with plasmid C3431(YEplac195-ASH1) and YCplac111, pRL200, pRL1206, pRL1207, pRL1138, or pRL1139. Transformants were grown in synthetic medium devoidof leucine and uracil. Subsequently, cells were processed for in situ hybridization using Cy3-conjugated cDNA probes for ASH1 mRNA.Representative images for in situ hybridization, DAPI staining, and Nomarski optics are shown. Also indicated is the percentage of cells withcrescent localized ASH1 mRNA and the percentage of cells exhibiting the full bud phenotype for ASH1 mRNA. The percentage of cells withlocalized ASH1 mRNA represents the average of two independent experiments with 50 cells counted in each experiment.



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mutants should also associate with ASH1 mRNA. We investi-gated whether She3p-S348E and She3p-S343E S361E associ-ate with ASH1 mRNA by immunoprecipitation and RT-PCR(Fig. 5A). We observed no RT-PCR product for ASH1 mRNAwhen the immunoprecipitation was performed with a cell ly-sate devoid of She3p (lane 1) or She2p (lane 2). We observedan RT-PCR product for ASH1 mRNA when the immunopre-cipitation was performed with cell lysates containing wild-typeShe3p-myc6 (lane 3), She3p-S348A-myc6 (lane 4), or She3p-S343A S361A-myc6 (lane 6). In contrast, we observed a sub-stantial reduction for the ASH1 mRNA RT-PCR productwhen the immunoprecipitation was performed with a cell ly-sate containing She3p-S348E-myc6 (lane 5) and no ASH1mRNA RT-PCR product when the immunoprecipitation wasperformed with a cell lysate containing She3p-S343E S361E-myc6 (lane 7). Given that She3p-S348E and She3p-S343ES361E associate with She2p in a manner indistinguishable fromthat of wild-type She3p, our results imply that the ability ofShe2p to tether ASH1 mRNA to She3p through a protein-protein interaction does not fully explain the ability of She3p toassociate with ASH1 mRNA.

Given that the buffer used for the immunoprecipitation/RT-PCR analysis (Fig. 5A) (25 mM HEPES-KOH [pH 7.5], 150

mM KCl, 2 mM MgCl2, 200 U of Superase-In [Ambion]/ml,0.1% NP-40, 1 mM dithiothreitol, 0.2 mg of heparin/ml, 0.1 �gof chymostatin/ml, 2 �g of aprotinin/ml, 1 �g of pepstatin/ml,0.5 �g of leupeptin/ml, 0.01 �g of benzamidine/ml) is differentfrom the buffer (50 mM HEPES-KOH [pH 7.3], 20 mM po-tassium acetate, 2 mM EDTA, 0.1% Triton X-100, 5% glyc-erol, 0.1 �g of chymostatin/ml, 2 �g of aprotinin/ml, 1 �g ofpepstatin/ml, 0.5 �g of leupeptin/ml, 0.01 �g of benzamidine/ml) used to monitor the association of She3p with Myo4p andShe2p (Fig. 3A and 4), we investigated whether She3p-S348Eand She3p-S343E S361E remain associated with She2p underthe immunoprecipitation conditions used for RT-PCR (Fig.5A). After immunoprecipitation with wild-type She3p-myc6

(lane 3), She3p-S348A-myc6 (lane 4), She3p-S348E-myc6 (lane5), She3p-S343A S361A-myc6 (lane 6), or She3p-S343ES361E-myc6 (lane 7), we observed that She2p was undetectableafter immunoprecipitation with any of these She3p alleles, andyet we detected the presence of ASH1 mRNA by RT-PCR withthe same immunoprecipitations. These results support our as-sertion that tethering of ASH1 mRNA to She3p through aprotein-protein interaction with She2p does not fully explainthe ability of She3p to associate with ASH1 mRNA.

To confirm the absence of the She2p-She3p interaction un-der the IP/RT-PCR conditions, we investigated whetherShe2p-myc6 could coimmunoprecipitate She3p and ASH1mRNA using the IP/RT-PCR conditions (Fig. 5B). Consistentwith our previous results, we observed that wild-type She3p(lane 3), She3p-S348A (lane 4), She3p-S348E (lane 5), She3p-S343A S361A (lane 6), and She3p-S343E S361E (lane 7) werenot detected after immunoprecipitation of She2p-myc6, and wedetected the presence of ASH1 mRNA by RT-PCR with thesame immunoprecipitations. Consequently, these results sup-port our findings that the conditions used for IP/RT-PCR areinadequate for the detection of the She2p-She3p interaction.

We further studied the She3p-ASH1 mRNA interaction bythree-hybrid analysis. As expected, She3p-S348A and She3p-S343A S361A express ASH1 mRNA-dependent LacZ activityat levels comparable to those of wild-type She3p (Fig. 5C).Consistent with our immunoprecipitation/RT-PCR analysis,we observed that She3p-S348E expresses ASH1 mRNA-depen-dent activity at lower levels than wild-type She3p. Unexpect-edly, we observed that She3p-S343E S361E expresses ASH1mRNA-dependent activity at levels comparable to wild-typeShe3p. The combination of the IP/RT-PCR results (Fig. 5A)with the three hybrid results (Fig. 5C) suggests that the She3p-S348E and She3p-S343E S361S mutants are differentially de-fective for association with ASH1 mRNA.

DISCUSSION

In this study we identify two novel mutants of She3p, S348Eand S343E S361E, which are defective for ASH1 mRNA lo-calization. Given that ASH1 mRNA localization requires thatShe3p associate with Myo4p and She2p, we investigatedwhether She3p-S348E and She3p-S343E S361E retain the abil-ity to associate with Myo4p and She2p. Since S348E and S343ES361E are located outside the domain of She3p that interactswith Myo4p, we did not expect that these amino acid substitu-tions would affect the She3p-Myo4p interaction (7). However,since both S348E and S343E S361E are located in the domain

TABLE 3. Summary for ASH1 mRNA localization in cellsexpressing the various She3p mutants

Amino acid(s)Localizationa

Ala Glu

S28 L LS217 L LS343 L LS348 L DS361 L LS392 L LT393 L LS394 L LS28, S343 L LS28, S361 L LS28, S392 L LS28, S394 L LS343, S361 L DS343, S392 L LS343, S394 L LS361, S392 L LS361, S394 L LS392, S394 L LS28, S343, S361 L DS28, S343, S392 L DS28, S343, S394 L LS28, S361, S394 L LS28, S392, S394 L LS343, S361, S392 L DS343, S361, S394 L DS343, S392, S394 L LS361, S392, S394 L LS28, S343, S361, S392 L DS28, S343, S361, S394 L DS28, S343, S392, S394 L DS28, S361, S392, S394 L DS343, S361, S392, S394 L DS28, S343, S361, S392, S394 L DS28, S217, S343, S348, S361, S392, S394, T393 L D

a L, localized; D, delocalized.



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of She3p that interacts with She2p, we anticipated that theseamino acid substitutions could affect the She3p-She2p interac-tion (7, 41). As expected, we determined that these She3pmutants retain the ability to associate with Myo4p but, unex-pectedly, we observed that these amino acid substitutions donot alter the She3p-She2p interaction. Although our assays,coimmunoprecipitation and two-hybrid analysis, indicate thatShe3p-S348E and She3p-S343E S361E associate with She2p atlevels indistinguishable from those of wild-type She3p, we can-not be certain that in vivo the She3p mutants have the sameaffinity for She2p as wild-type She3p. Furthermore, if wild-typeShe3p induces a conformation change in She2p, She3p-S348Eand She3p-S343E S361E might be defective for inducing theconformation change, while retaining the ability to associatewith She2p. In any event, our results suggest that the ability ofShe3p to associate with Myo4p and She2p does not fully ac-count for the function of She3p in ASH1 mRNA localization.

To begin to identify a novel function for She3p in ASH1mRNA localization, we investigated whether She3p-S348E andShe3p-S343E S361E can associate with ASH1 mRNA. Basedon the current model for ASH1 mRNA localization, She3p istethered to ASH1 mRNA through a protein-protein interac-tion with She2p (7, 41, 63). Since the She3p mutants retain theability to associate with She2p, the current model for ASH1mRNA localization predicts that the She3p mutants shouldretain the ability to associate with ASH1 mRNA. However, wedetermined that She3p-S348E and possibly She3p-S343ES361E are defective for association with ASH1 mRNA. This

FIG. 4. She3p-S348E and She3p-S343E S361E retain the ability tosimultaneously associate with Myo4p and She2p. Yeast strainYLM1406 was transformed with the following combinations of plas-mids: pRL519 (She3p-wt)/pRL1143 (She2p-wt) (lane 1), YCplac111/pRL1143 (lane 2), pRL519/YCplac33 (lane 3), pRL519/pRL1143 (lane4), pRL1252 (She3p-S348A)/pRL1143 (lane 5), pRL1253 (She3p-S348E)/pRL1143 (lane 6), pRL1182 (She3p-S343A S361A)/pRL1143(lane 7), and pRL1183 (She3p-S343E S361E)/pRL1143 (lane 8).Transformants were grown in medium lacking leucine and uracil, andcell lysates were prepared. Myo4p-myc13 was immunoprecipitatedfrom the lysate with an anti-myc monoclonal antibody (lanes 2 to 8) orfrom a no-antibody control (lane 1). Subsequently, whole-cell lysatesand immunoprecipitations were analyzed by Western blotting for thepresence of Myo4p-myc13, She3p, and She2p with anti-myc, rabbitpolyclonal She3p antiserum and rabbit polyclonal She2p antiserum,respectively.

FIG. 3. She3p-S348E and She3p-S343E S361E retain the ability to associate with Myo4p and She2p. (A) She3p-S348E and She3p-S343E S361Ecoimmunoprecipitate Myo4p and She2p. Yeast strain YLM1320 was transformed with the following combinations of plasmids: YCplac111/pRL1143 (She2p-wt) (lane 1), pRL200 (She3p-wt-myc6)/YCplac33 (lane 2), pRL200/pRL1143 (lane 3), pRL1206 (She3p-S348A-myc6)/pRL1143(lane 4), pRL1207 (She3p-S348E-myc6)/pRL1143 (lane 5), pRL1138 (She3p-S343A S361A-myc6)/pRL1143 (lane 6), and pRL1139 (She3p-S343ES361E-myc6)/pRL1143 (lane 7). Transformants were grown in medium lacking leucine and uracil, and cell lysates were prepared. She3p-myc6 wasimmunoprecipitated from the lysate with anti-myc monoclonal antibody. Subsequently, the whole-cell lysates and immunoprecipitations wereanalyzed by Western blotting for the presence of She3p-myc6, Myo4p-3HA, and She2p with rabbit polyclonal She3p antiserum, anti-HA, and rabbitpolyclonal She2p antiserum, respectively. (B) She3p-S348E and She3p-S343E S361E are able to associate with She2p in the two-hybrid assay. Yeaststrain PJ69-4a was transformed with the following combinations of plasmids: pGBDU-c2 (vector)/pACT2 (vector), pGBDU-c2/pEP13 (pACT2-She3p-wt), pRL174 (pGBDU-c2-She2p-wt)/pACT2, pRL174/pEP13, pRL174/pRL1225 (pACT2-She3p-S348A), pRL174/pRL1226 (pACT2-She3p-S348E), pRL174/pRL1221 (pACT2-She3p-S343A S361A), or pRL174/pRL1222 (pACT2-She3p-S343E S361E). Transformants were grownin liquid culture and processed for �-galactosidase assays.



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result implies that tethering of ASH1 mRNA through She2pdoes not fully account for the ability of She3p to associate withASH1 mRNA. Although it has been previously demonstratedthat She2p is required for the association of ASH1 mRNA withShe3p, our results showing that the She2p-She3p interaction isnot maintained under the conditions used to perform IP/RT-

PCR imply that She2p may be necessary to establish the asso-ciation between ASH1 mRNA and She3p, but She2p may notbe necessary to maintain the interaction between ASH1mRNA and She3p (7, 41).

Based on the IP/RT-PCR data, one might anticipate lowerLacZ activity in the three-hybrid assay for She3p-S348E and

FIG. 5. She3p-S348E and She3p-S343E S361E are defective for association with ASH1 mRNA. (A) Immunoprecipitation of various She3p-myc6 alleles, followed by the detection of She2p and ASH1 mRNA by Western blot and RT-PCR, respectively. Yeast strain YLM1320 wastransformed with the following combinations of plasmids: YCplac111/pRL1143 (She2p-wt) (lane 1), pRL200 (She3p-wt-myc6)/YCplac33 (lane 2),pRL200/pRL1143 (lane 3), pRL1206 (She3p-S348A-myc6)/pRL1143 (lane 4), pRL1207 (She3p-S348E-myc6)/pRL1143 (lane 5), pRL1138 (She3p-S343A S361A-myc6)/pRL1143 (lane 6), and pRL1139 (She3p-S343E S361E-myc6)/pRL1143 (lane 7). Transformants were grown in mediumlacking leucine and uracil, and cell lysates were prepared. She3p-myc6 was immunoprecipitated from the lysate with anti-myc monoclonal antibody.Subsequently, the whole-cell lysates and immunoprecipitations were used for RT-PCRs with primers specific for ASH1 mRNA in the absence (�)and presence (�) of RT. In addition, the total cell lysates and immunoprecipitations were analyzed by Western blotting for She3p-myc6 and She2pwith rabbit polyclonal She3p antiserum and rabbit polyclonal She2p antiserum, respectively. (B) Immunoprecipitation of wild-type She2p-myc6,followed by the detection of various She3p alleles and ASH1 mRNA by Western blot and RT-PCR, respectively. Yeast strain YLM1320 wastransformed with the following combinations of plasmids: pRL676 (She2p-myc6)/YCplac111 (lane 1), YCplac22/pRL519 (She3p-wt) (lane 2),pRL676/pRL519 (lane 3), pRL676/pRL1252 (She3p-S348A) (lane 4), pRL676/pRL1253 (She3p-S348E) (lane 5), pRL676/pRL1182 (She3p-S343AS361A) (lane 6), and pRL676/pRL1183 (She3p-S343E S361E) (lane 7). Transformants were grown in medium lacking leucine and tryptophan andcell lysates prepared. She2p-myc6 was immunoprecipitated from the lysate with anti-myc monoclonal antibody. Subsequently, the whole-cell lysatesand immunoprecipitations were used for RT-PCRs with primers specific for ASH1 mRNA in the absence (�) or presence (�) of RT. In addition,the total cell lysates and immunoprecipitations were analyzed by Western blotting for She2p-myc6 and She3p with rabbit polyclonal She2p andrabbit polyclonal She3p antiserum, respectively. (C) Investigation of the ability of She3p mutants to associate with ASH1 mRNA by using thethree-hybrid assay. Yeast strain YML584 was transformed with the following combinations of plasmids: pIIIA-MS2-2 (vector)/pACT2 (vector),pRL80 (pIIIA/MS2-2-E3)/pACT2, pIIIA/MS2-2/pEP13 (She3p-wt), pRL80/pEP13 (She3p-wt), pRL80/pRL1225 (She3p-S348A), pRL80/pRL1226(She3p-S348E), pRL80/pRL1221 (She3p-S343A S361A), or pRL80/pRL1222 (She3p-S343E S361E). Transformants were grown in liquid cultureand processed for the �-galactosidase assay.



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She3p-S343E S361E. However, our data imply that She3p canassociate with ASH1 mRNA through multiple, distinct inter-actions. For She3p-S348E and She3p-S343E S361E, some ofthese interactions are maintained in the three-hybrid assay,while the same interactions are not maintained in the IP/RT-PCR assay. Consequently, the results from these two assaysmay not entirely coincide. Furthermore, we observed a differ-ence between She3p-S348E and She3p-S343E S361E in thethree-hybrid assay. For She3p S348E, we observed a reductionin the association with ASH1 mRNA by the IP/RT-PCR assayand the three-hybrid assay, whereas for She3p-S343E S361Ewe observed a defect for the association with ASH1 mRNAwith the IP/RT-PCR experiment but not with the three-hybridexperiment. These results suggest that the two She3p mutantsdifferentially associate with ASH1 mRNA. We hypothesizethat She3p-S348E is defective for association with individualcis-acting localization elements as determined by the IP/RT-PCR and three-hybrid assays. In contrast, She3p-S343E S361Emight only be defective for the association with full-lengthASH1 mRNA, which is limited to detection in the IP/RT-PCRassay. In any event, our results clearly show that She3p-S348Eand She3p-S343E S361E are defective for ASH1 mRNA local-ization through a novel activity, since these mutants retain theability to associate with Myo4p and She2p.

Previously, we demonstrated that a reporter mRNA artifi-cially tethered to She2p could not localize in a manner analo-gous to ASH1 mRNA, and these results implied that mRNAartificially tethered to She2p could not properly anchor at thebud tip (24). From these results we hypothesized that a mo-lecular reorganization event occurs that releases She2p fromthe Myo4p-She3p-ASH1 mRNA anchoring complex. Our re-sults with She3p-S348E and She3p-S343E S361E support ourprevious hypothesis that a Myo4p-She3p-ASH1 mRNA com-plex devoid of She2p does exist. However, our results do notdistinguish between at least two possible models. In the firstmodel, She2p could identify ASH1 mRNA in the nucleus and

escort ASH1 mRNA to the cytoplasm, where She2p functionsto directly load ASH1 mRNA onto She3p (Fig. 6A). Thismodel necessitates that She3p possess intrinsic RNA-bindingactivity, and further suggests that no additional proteins maybe present in the Myo4p-She3p-ASH1 mRNA complex. How-ever, homology searches reveal no obvious RNA-binding mo-tifs within She3p. Consequently, if She3p directly contactsASH1 mRNA, the She3p-ASH1 mRNA association most likelyoccurs through a novel RNA-binding domain.

In the second model She2p could identify ASH1 mRNA inthe nucleus and escort ASH1 mRNA to the cytoplasm whereShe2p functions to directly load ASH1 mRNA onto an uniden-tified RNA-binding protein that directly associates with She3p(Fig. 6B). This model predicts that the protein-protein inter-action between She3p and the unidentified RNA-binding pro-tein would be stable under the IP/RT-PCR conditions, unlikethe She2p-She3p interaction. Furthermore, this model predictsthat additional novel proteins remain to be identified in theMyo4p-She3p-ASH1 mRNA complex. Global two-hybrid andaffinity purification experiments have determined that She3passociates with at least 28 proteins, and the possible role for 23of these proteins in ASH1 mRNA localization remains to bedetermined (14, 17, 20, 21, 31, 38, 46, 49, 52, 55, 64–67, 69).However, none of the 23 proteins appear to be RNA-bindingproteins. Consequently, if any of the 23 proteins have a role inASH1 mRNA localization, it would be interesting to determinewhether these proteins possess RNA-binding activity.

We initiated the studies presented here to address the hy-pothesis that phosphorylation of She3p regulates ASH1mRNA localization activities. Global mass spectrometry ap-proaches have determined that Ser 343, 348, and 361 are sitesof phosphorylation (1, 27, 40), and our studies demonstratingthat She3p-S348E and She3p-S343E S361E, which mimic con-stitutive phosphorylation, are defective for ASH1 mRNA lo-calization support the hypothesis that phosphorylation ofShe3p negatively regulates a novel ASH1 mRNA localization

FIG. 6. Two models for the association of ASH1 mRNA with She3p. (A) She2p identifies ASH1 mRNA in the nucleus. Once the She2p-ASH1mRNA complex reaches the cytoplasm, She2p directly loads ASH1 mRNA onto She3p. She2p could load ASH1 mRNA onto a She3p transportcomplex or a She3p anchoring complex (AC). (B) She2p identifies ASH1 mRNA in the nucleus. Once the She2p-ASH1 mRNA complex reachesthe cytoplasm, She2p loads ASH1 mRNA onto an unidentified RNA-binding protein (X) that indirectly tethers ASH1 mRNA to She3p. Onceagain, She2p could load ASH1 mRNA onto either a transport complex and/or an anchoring complex (AC).



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activity. If She3p possesses intrinsic RNA-binding activity,phosphorylation of She3p at Ser 343, 348, and 361 could neg-atively regulate She3p RNA-binding activity. There is prece-dent for RNA-binding activity being negatively regulated byphosphorylation. Khd1p and Puf6p are RNA-binding proteinsthat function to translationally repress ASH1 mRNA until itreaches the bud, and the RNA-binding activities for Khd1pand Puf6p are negatively regulated by phosphorylation (16,50). Furthermore, the RNA-binding activity for Zbp1p, whichis involved in the localization of �-actin mRNA in chickenembryo fibroblasts, is also negatively regulated by phosphory-lation (32). However, if She3p indirectly associates with ASH1mRNA through another unidentified RNA-binding protein,this She3p protein-protein interaction could be negatively reg-ulated by phosphorylation. Analogous to RNA-binding activ-ity, there are numerous examples of protein-protein interac-tions being negatively regulated by phosphorylation.

If phosphorylation at Ser 343, 348, and 361 is regulatingShe3p ASH1 mRNA localization activities, one might haveexpected that alanine substitutions at these amino acids wouldadversely affect ASH1 mRNA localization. However, our find-ings that alanine substitutions at these amino acids retainASH1 mRNA localization activity support the hypothesis thatphosphorylation at Ser 343, 348, and 361 is not required forASH1 mRNA localization. Furthermore, other phosphopro-teins exhibit a similar phenotype. In S. cerevisiae, the activityfor iron regulatory protein 1 (Irp1p) is regulated by phosphory-lation at Ser 138. Analogous to She3p, the Irp1p-S138A mu-tant exhibits wild-type activity, while the activity for the S138Emutant is substantially reduced compared to wild-type Irp1p(8). It is particularly intriguing that the She3p mutant contain-ing Ala at all of the Ser and Thr sites of phosphorylation isactive for ASH1 mRNA localization. If phosphorylation ofShe3p is required for ASH1 mRNA localization, possibly ad-ditional redundant sites need to be altered to observe an effecton ASH1 mRNA localization. Since She3p contains 69 Serresidues and 26 Thr residues, it remains a distinct possibilitythat additional sites of phosphorylation remain to be identified.

Global kinase studies indicate that She3p can be phosphory-lated in vitro by at least 13 yeast kinases: Atg1p, Hsl1p, Ime2p,Ksp1p, Pho85p Pcl1p, Prk1p, Rad53p, Snf1p, Ste20p, Swe1p,Tpk1p, Yck1p, and Yck2p (52). Using the yeast deletion col-lection we have examined if the absence of Atg1p, Hsl1p,Ime2p, Ksp1p, Pho85p Pcl1p, Prk1p, Snf1p, Ste20p, Swe1p,Tpk1p, or Yck2p delocalize ASH1 mRNA and, consistent withour analyses of serine-to-alanine amino acid substitutions inShe3p, we observed no evidence that phosphorylation of She3pis necessary for ASH1 mRNA localization. Based on our re-sults with She3p-S348E and S343E S361E, we would predictthat the absence of the phosphatases responsible for removingthe phosphate group from Ser 343, 348, and 361 should exhibita defect in ASH1 mRNA localization. However, the identity ofcandidate phosphatases remains elusive.

In conclusion, our studies provide compelling evidence thatShe3p possesses a novel function required for ASH1 mRNAlocalization and imply that the novel function is related to theability of She3p to associate with ASH1 mRNA. Furthermore,our results suggest that phosphorylation could negatively reg-ulate the novel She3p-RNA localization activity. In the futurethe identification and characterization of the novel She3p-

RNA localization activity will provide new insight into themechanism responsible for ASH1 mRNA localization.

ACKNOWLEDGMENTS

We thank Amy Hudson’s laboratory for generously providing theHA antibody. We also thank Claudia Kale for helpful discussions andcritically reading the manuscript.

This study was supported by NIH grant GM60392 and the PewScholars Program in the Biomedical Sciences.

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She3p Possesses a Novel Activity Required for ASH1 mRNA … · ASH1 mRNA is proposed to be statically anchored through a mechanism requiring translation of ASH1 mRNA (5, 26, 33)