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Originally published In Press as doi:10.1074/jbc.M402068200 on June 10, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36962-36971, August 27, 2004
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Involvement of the Late Secretory Pathway in Actin Regulation and mRNA Transport in Yeast*

Stella Aronov{ddagger} and Jeffrey E. Gerst§

From the Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

Received for publication, February 25, 2004 , and in revised form, June 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Both the delivery of secretory vesicles and asymmetric distribution of mRNA to the bud are dependent upon the actin cytoskeleton in yeast. Here we examined whether components of the exocytic apparatus play a role in mRNA transport. By screening secretion mutants in situ and in vivo, we found that all had an altered pattern of ASH1 mRNA localization. These included alleles of CDC42 and RHO3 (cdc42-6 and rho3-V51) thought to regulate specifically the fusion of secretory vesicles but were found to affect strongly the cytoskeleton as well. Most interestingly, mutations in late secretion-related genes not directly involved in actin regulation also showed substantial alterations in ASH1 mRNA distribution. These included mutations in genes encoding components of the exocyst (SEC10 and SEC15), SNARE regulatory proteins (SEC1, SEC4, and SRO7), SNAREs (SEC9 and SSO1/2), and proteins involved in Golgi export (PIK1 and YPT31/32). Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport. Our novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The establishment of cell polarity in eukaryotes involves the asymmetric organization of mRNA, the cytoskeleton, and the secretory pathway to lead to the polarized distribution of new membrane along a given axis (1, 2). In yeast, polarization leads to the budding of daughter cells (cell division), the asymmetric segregation of cell-fate determinants, and mating of haplotypes (3-4). These aspects of polarization require proper control of the actin cytoskeleton, as mutations therein block the exocytosis of proteins and new membrane along the axis of growth as well as the delivery of an mRNA encoding a protein involved in mating-type control (e.g. Ash1). For example, loss-of-function mutations in genes encoding yeast actin (ACT1), tropomyosin (TPM1,2), and a type V myosin (MYO2) all block exocytosis and result in lethality (5-7). Similarly, mutations in actin, tropomyosin, and another type V myosin (encoded by MYO4) also block ASH1 mRNA transport (2, 8-11). Thus, an essentially common mechanism for both vesicle and mRNA transport to the growing bud appears to have evolved in eukaryotes.

A key question that remains to be resolved is whether components of the secretory apparatus play a role in mRNA transport and localization to the bud. Because both share an actin-dependent transport mechanism, it seems likely that feedback from the exocytic pathway may affect actin assembly and, therefore, subsequent mRNA transport. In yeast, SHE4, one of five SHE genes known to be required for the transport and localization of ASH1 mRNA (2, 9-12), was isolated as an endocytosis-defective mutant and was shown to have a depolarized actin cytoskeleton (13). Thus, components of the endocytic apparatus that regulate actin may also influence mRNA transport. Until recently, the function of She4 in either endocytosis or actin regulation was not known. However, it was shown that She4 interacts directly with the motor domain of unconventional myosins, including Myo4/She1, and that mutations in this domain can bypass she4{Delta} growth and endocytosis defects but not defects in mating-type switching (14). This suggests that She4 may serve multiple roles, one in the regulation of myosin motor function and perhaps an additional role in ASH1 mRNA transport (14). These are likely to be mediated by different domains of the protein, as expression of the C-terminal domain of She4 suppresses the growth defects in the she4{Delta} mutant, whereas full-length She4 is necessary to suppress both the growth and the HO gene expression phenotypes (14). Other endocytic proteins have been shown to modulate mRNA transport and localization, including Drosophila Rab11 which plays a role in endocytic protein sorting, microtubule organization, and also oskar mRNA localization (15). Thus, a connection between endocytic transport, regulation of the microtubule cytoskeleton, and mRNA localization exists in higher organisms and may parallel that observed in yeast.

Here we have examined directly whether mutations in components of the late secretory pathway affect the transport of ASH1 mRNA to the daughter cell and its localization therein using both in situ and in vivo assays. Most importantly, we have found that both polarity and secretion-related proteins modulate the asymmetric distribution of ASH1 mRNA. For example, alleles of small GTPases (e.g. cdc42-6 and rho3-V51) thought to mediate specifically secretory vesicle fusion and not to affect significantly the actin cytoskeleton (16, 17) greatly inhibited ASH1 distribution to the bud. This effect was also observed in yeast bearing mutations in either RAS2 (e.g. ras2{Delta}) or CMD1 (e.g. cmd1-239), although we note that the actin cytoskeleton was substantially disorganized in all of these mutants.

Most surprisingly, however, conditional loss-of-function mutations in genes thought to be directly involved in protein export, and not actin regulation, revealed numerous cases whereby mRNA localization was also affected. For example, mutations in genes encoding proteins involved in the transport and fusion of secretory vesicles, like Sec4 and Sro7 (18, 19) as well as Pik1, a phosphatidylinositol 4-kinase involved in Golgi export (20, 21), all greatly inhibited ASH1 mRNA distribution to the bud. To a somewhat lesser extent, mutations in the Sec1 SNARE1 regulator (22), the exocyst components, Sec10 and Sec15 (23) (the latter being a possible Sec4 effector (24)), and the exocytic SNAREs, Sec9, and Sso1/2 (25, 26), also resulted in the mislocalization of ASH1 mRNA. This suggests that SNAREs, SNARE regulatory, exocyst, and Golgi export proteins all exert control over mRNA transport and localization. Subsequent analysis of the actin cytoskeleton in these mutants revealed that all have a disorganized pattern of actin labeling, which occurs rapidly upon the shift to semi-restrictive and restrictive temperatures. This indicates that a novel relationship exists between vesicle transport and the integrity of the actin cytoskeleton. Thus, active exocytosis in yeast is necessary for both maintenance of the polarized actin cytoskeleton and subsequent mRNA transport and localization to the growing bud.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Media, DNA, and Genetic Manipulations—Yeast were grown in standard growth media containing 2% glucose. Synthetic complete and drop-out media were prepared similar to that described previously (27). Standard methods were used for the introduction of DNA into yeast and the preparation of genomic DNA (27). Cells transformed with plasmids for ASH1 mRNA labeling (see below) were grown on selective synthetic medium. For the induction of MS2 coat protein fused to GFP (MS2-GFP), cells were switched to synthetic medium lacking methionine for 1-2 h.

Yeast Strains—Yeast strains used are listed in Table I. Plasmids—Plasmids encoding an ASH1 gene fragment with an MS2 viral coat protein-binding site in its 3'-untranslated region (pIIIA/ASH1-UTR) and a gene fusion of the MS2 coat protein and GFP (pCPGFP) (28) were generously provided by Kerry Bloom (University of North Carolina, Chapel Hill).


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  		TABLE I
Yeast strains

 
Plasmids bearing genomic ASH1 were created for this study. The coding region of the ASH1 was amplified by PCR from genomic DNA using forward (Ash1-For, 5'-CCTATCGCTCCTGTCCTATCCTTATTACG-3') and reverse (Ash1-Rev, 5'-AGTTATTAGTTGAAAGAGCTCCAGTTATCC-3') oligonucleotides. The 1751-bp fragment was cloned into pGEM-T vector (Promega, Madison, WI) to yield pGEM-ASH1. For creation of the DNA template used to make the RNA probe for in situ hybridization, a 250-bp fragment of the ASH1 coding region was obtained by the digestion of pGEM-ASH1 with SphI and NdeI. This fragment was then ligated into the SphI and NdeI sites of pGEM to yield pGEM-ASH1short.

Fluorescence in Situ Hybridization (FISH) and Immunofluorescence—ASH1 mRNA was detected by FISH with the following modifications. Cultures were grown to A600 = 0.5, fixed in 4% formaldehyde for 1 h, washed, and then spheroplasted in 100 mM potassium phosphate buffer, pH 6.5, containing 1.2 M sorbitol, 30 mM {beta}-mercaptoethanol, and 40 µg/ml zymolase (100T; ICN Biomedicals, Aurora, OH) for 30 min at 24 °C. Spheroplasts were washed and spread onto clean polylysine-coated, multiwell test slides (ICN Biomedicals). Cells were incubated for 1 h at 50 °C in hybridization mix (50% formamide, 5x SSC, yeast tRNA (1 mg/ml), heparin (100 µg/ml), 1x Denhardt's solution, 0.1% Tween 20, 0.1% CHAPS, and 5 mM EDTA) and then incubated overnight at 50 °C in hybridization mix containing 5 µg/ml digoxigenin-labeled 250-bp ASH1 RNA probes. ASH1 RNA probes were synthesized in both the sense and antisense orientations, using the digoxigenin RNA labeling kit (Roche Applied Science) according to the manufacturer's protocol. After hybridization, cells were washed in 0.2x SSC and blocked in 1x PBS containing 0.1% Triton X-100 and 10% horse serum. Cells were incubated with horseradish peroxidase-conjugated anti-digoxigenin monoclonal antibodies (1:500) (Jackson ImmunoResearch, West Grove, PA) in blocking buffer for 2 h. After washing, cells were incubated with Cy5-conjugated anti-horseradish peroxidase antibodies (1:100) (Jackson ImmunoResearch) for 2 h at room temperature. Cells were then washed and mounted in 0.01 M Tris-Cl, pH 8.4, 90% glycerol, 1 mg/ml p-phenylenediamine, and 0.1 µg/ml propidium iodide (Sigma).

The actin cytoskeleton was stained with rhodamine-conjugated phalloidin (Sigma). Cells were grown to A600 = 0.5 in YPD and fixed with formaldehyde at a final concentration of 4% for 1 h. Cells were harvested, washed twice with 1x PBS, permeabilized using 0.5% Nonidet P-40 in PBS for 5 min, and washed again with 1x PBS. For staining, 100-µl aliquots of cells were incubated with rhodamine-conjugated phalloidin (final concentration of 0.165 µM) for 15-60 min on ice and washed with 1x PBS. Cells were mixed with mounting medium and mounted on glass slides with coverslips prior to visualization. To obtain quantitative data on the organization of the actin cytoskeleton, between 50 and 100 cells of each strain were scored for the distribution of actin patches in the mother and bud. Data are summarized in Table V.


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  		TABLE V
Actin organization in yeast secretion and secretion-related mutants

 
Image Analysis—Fluorescence imaging was performed using a Zeiss LSM confocal microscope with LSM510 software equipped with x40 and x100 oil immersion lenses. The following wavelengths were used: rhodamine-phalloidin (excitation 545 nm and emission 560-580 nm); GFP (excitation 480 nm and emission 530 nm), and Cy5 (excitation 650 nm and emission 680 nm). Control experiments for in situ hybridization showed no observable background using the sense ASH1 RNA probe. Likewise, no signal was observed in the absence of primary antibodies.

For live cell imaging, cells were grown overnight to log phase in synthetic medium at 26 °C and shifted to synthetic medium lacking methionine for 1 h to induce the production of coat protein-GFP. For temperature-shift experiments, cells were incubated in medium lacking methionine at the appropriate restrictive temperature. Cells were concentrated, supplemented with gelatin (Sigma) or low melting point agarose (FMC, Rockland, ME) (0.25 or 0.5% final concentration, respectively), and mounted on glass slides or coverslips. A temperature-controlled chamber (Warner Instrument Corp.) was used to maintain cells at constant temperature. Optical sectioning was performed as described in Shaw and Quatrano (29). Cells were sectioned optically at 0.75-µm increments for a total of 3.0 µm. Measurement of mRNA Localization and Distribution in Situ—To obtain quantitative data on the localization of the ASH1 mRNA in each strain, between 30 and 80 cells with visible buds (i.e. cells in S and G2/M phase) were scored for a localized or mislocalized ASH1 mRNA signal using FISH (note: ASH1 mRNA localizes to the newly forming bud tip in early S phase). Thus, an ASH1 mRNA signal was considered as localized when it was present at the bud tip or localized along one side of the bud. An mRNA signal was considered as mislocalized to the bud and mother when it was distributed in both the bud and mother cell (i.e. transported to but not anchored at the bud tip). An mRNA signal was considered mislocalized to the mother when it was found in the mother cell only (neither transported to nor anchored in the bud). The standard deviation obtained from two separate counts (per strain) was calculated. The data obtained are summarized in Tables II, III, IV.


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  		TABLE II
In situ ASH1 mRNA localization in small GTPase mutants

 


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  		TABLE III
In situ ASH1 mRNA localization in SNARE, SNARE regulator, and exocyst mutants

 


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  		TABLE IV
In situ ASH1 mRNA localization in cytoskeleton and other mutants

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In Situ Localization of ASH1 mRNA in Small GTPase Mutants of the Late Secretory Pathway—ASH1 mRNA localization to the bud tip has been shown to be dependent upon proteins involved in mRNA binding and transport along the actin cytoskeleton (2, 8-12). The latter include actin, tropomyosins 1/2, and myosin 4, and these are encoded by ACT1, TPM1/2, and MYO4, respectively. To determine the involvement of known secretory proteins, we employed fluorescence-based in situ hybridization (FISH) by using digoxigenin-labeled sense and antisense ASH1 probes and mutants of the late secretory pathway in yeast. We first examined whether temperature-sensitive mutations in small GTPases may influence the localization of ASH1 mRNA after shifting cells to restrictive temperatures (see Table II and Fig. 1A).



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  		FIG. 1.
In situ localization of ASH1 mRNA in mutants of the late secretory pathway. A, in situ localization of ASH1 mRNA in control cells and small GTPase mutants. All yeasts were grown to log phase on rich medium prior to fixation and in situ hybridization with a digoxigenin-labeled ASH1 probe (see "Experimental Procedures"). Yeasts were either maintained at 26 °C or shifted to other temperatures (sec4-8, 1 h at 37 °C; rho3-V51, 1 h at 15 °C; cdc42-6, 1 h at 33 °C; wild-type (WT) cells, 1 h at 15 °C or 37 °C) prior to fixation. The panels show nuclear staining performed with propidium iodide (Nuc); ASH1 mRNA visualized using CY-conjugated anti-digoxigenin antibodies (mRNA); and merger of the Nuc/mRNA windows with Nomarski visualized cells (Merge/Nom). B, in situ localization of ASH1 mRNA in exocyst, SNARE regulator, and SNARE mutants. Yeast were grown to log phase and either maintained at 26 °C or shifted to restrictive temperatures (temp) (sro7{Delta}/77{Delta}, 1 h at 18 °C; sso2-1 and sec1-1, 1 h at 37 °C), prior to fixation and in situ hybridization. C, in situ localization of ASH1 mRNA in yeast actin cytoskeleton and other mutants. Yeast were grown to log phase and either maintained at 26 °C or shifted to elevated temperatures (pik1-83, 1 h at 37 °C; tpm1-2 tpm2{Delta}, 1 h at 37 °C; cmd1-239, 1 h at 37 °C), prior to fixation and in situ hybridization.

 
ASH1 mRNA localization was examined in budded S and G2/M phase yeast cells by using FISH, and the results were statistically compiled from 30 to 80 cells per condition. Specific FISH signals were observed by using the antisense probes only.2 We found that >75% of wild-type cells had ASH1 mRNA present at the bud tip (Table II and note examples in Fig. 1A), as shown previously (8, 9), and did not vary significantly with temperature (15-37 °C). Nonetheless, a small number of cells (<20%) were found to have some mRNA located in the nucleus of the mother cell, the latter detected by propidium iodide staining, which probably represents a maternal pool of ASH1 mRNA that had yet to be exported and delivered to the daughter cell. In contrast to wild-type cells, less than 20% of myo4{Delta} deletion mutants had ASH1 mRNA localized at the bud tip (Table IV and Fig. 1A). Thus, myo4{Delta} mutants are defective in ASH1 mRNA localization, as determined previously (8, 9). Most interestingly, we found that a temperature-sensitive mutation in Cdc42 (cdc42-6), which was proposed to act only upon vesicle transport at 33 °C and not to affect the actin cytoskeleton (17), had prominent effects upon ASH1 mRNA localization at either permissive (26 °C) or restrictive (33 °C) temperatures (Table II and Fig. 1A). At the permissive temperature about 50% of the mRNA was mislocalized in the bud or to the mother cell, whereas 70% was mislocalized at the restrictive temperature. Moreover, nearly identical results were obtained with a temperature-sensitive mutant of the Cdc42 exchange factor, Cdc24 (cdc24-1), which showed similar amounts of mislocalized ASH1 mRNA overall (Table II). Thus, Cdc42 is likely to play a significant role in the delivery of ASH1 to the daughter cell.

Next, we examined other GTPases (Table II) including the following: Sec4, which confers secretory vesicle tethering and SNARE assembly (30); Rho3, which regulates actin assembly but may also play a role in vesicle transport (16); Ypt31/32, which is involved in Golgi export (31); and Ras2, which regulates signaling pathways involved in both cell growth and actin regulation (32). Similar to cdc42-6, we found that an allele of Rho3 (rho3-V51) that was proposed to act upon vesicle transport alone (16) had strong effects upon ASH1 mRNA localization (Table II). At the permissive temperature (26 °C) about 40% of the cells had mislocalized ASH1 mRNA, whereas this increased to 85% at the restrictive temperature (15 °C; 1 h), wherein most of the mRNA accumulated exclusively in the mother cell. Interestingly, ASH1 mRNA was mislocalized at restrictive temperatures with all of the other GTPase mutants examined, including the following: sec4-8 (37 °C; 1 h) (for example, see Fig. 1A); sec4-P48 (15 °C; 1 h); ypt31{Delta} ypt32-1 (37 °C; 1 h); and ras2{Delta} cells (37 °C; 1 h). However, in these particular strains ASH1 mRNA was distributed in both mother and daughter cells and was not restricted to the mother cell as seen in cdc42-6 and rho3-V51 cells. Nonetheless, the finding that two different alleles of SEC4, which encodes a protein involved in the terminal steps of vesicle docking and fusion, affect ASH1 mRNA localization strongly suggested a direct involvement of the secretory pathway in mRNA transport and localization.

In Situ Localization of ASH1 mRNA in Exocyst, SNARE Regulator, and SNARE Mutants of the Late Secretory Pathway—We next examined ASH1 mRNA localization in situ in mutants of proteins directly involved in vesicle tethering, docking, and fusion (Table III and Fig. 1B). These include the following exocyst components: Sec10 and Sec15 (23); the following SNARE regulators: Sec1 (22), Sro7, and Sro77 (19); and the Sso1/2 t-SNAREs (26). We found that a deletion mutation in the yeast tomosyn homolog, Sro7, resulted in ASH1 mRNA mislocalization in the bud and to the mother cell in over 50% of the cells examined at 37 °C, whereas a similar mutation in its homolog, Sro77, had little to no effect (Table III).2 A sro7{Delta} sro77{Delta} double mutant had a somewhat stronger phenotype than sro7{Delta}, resulting in 80% of cells having ASH1 mRNA co-mislocalized in the mother and bud and 20% mislocalized to the mother alone at the restrictive temperature (Table III and see Fig. 1B for example).

A temperature-sensitive mutation in the yeast SM protein, Sec1, also had a significant effect upon ASH1 mRNA localization and resulted in mislocalization in both mother and bud in nearly 50% of the cells examined at 37 °C (Table III and Fig. 1B). Thus, both the Sro7 and Sec1 SNARE regulators affect ASH1 mRNA transport. Similarly, a temperature-sensitive mutation in the yeast syntaxin t-SNARE, Sso2, also had an effect upon ASH1 mRNA localization, resulting in mislocalization to both the mother and bud, but only in about 40% of the cells (Table III and Fig. 1B).

Next, we examined localization in two of the eight exocyst components and found that a mutation in the Sec4 effector, Sec15 (24), had a prominent effect resulting in the exclusive localization of ASH1 mRNA to the mother cells in 15% of the yeast at the restrictive temperature. This effect was actually somewhat stronger than that observed for the sec4 mutants (see Table II). However, only ~40% of the cells had mRNA mislocalized to both mother and bud in either the Sec10 or Sec15 mutants at the restrictive temperature, whereas 60-70% co-mislocalization was observed for the sec4 mutants (Tables II and III). Together, these results suggest that ASH1 mRNA transport and localization are strongly influenced by proteins acting at the level of vesicle docking and fusion.

In Situ Localization of ASH1 mRNA in Yeast Actin Cytoskeleton and Other Mutants—Finally, we examined the localization of ASH1 mRNA in situ in actin cytoskeleton mutants known to be defective for transport, as controls, and in a few other mutants. As expected, ASH1 mRNA was strongly mislocalized to the mother and co-mislocalized in the mother-bud in tpm1{Delta} and tpm1-2 tpm2{Delta} cells at the restrictive temperature and in myo4{Delta} cells at 26 °C (Table IV and Fig. 1C). Under these conditions, this resulted in nearly 80% of the cells having mislocalized ASH1 mRNA, as shown earlier (8, 9). We also found that myo2-66 cells, which bear a temperature-sensitive mutation in a type V myosin required for vesicular transport and vacuolar inheritance (7), had a milder mislocalization effect at 37 °C (Table IV) similar to that shown previously (8). Interestingly, we found that two other trafficking mutants had dramatic effects upon ASH1 mRNA localization. A temperature-sensitive mutation in PIK1, which encodes a phosphatidylinositol 4-kinase involved in Golgi export (21), resulted in significant co-mislocalization (~70%) in the mother and bud at the restrictive temperature (Table IV and Fig. 1C). A combination between an arf1{Delta} deletion and the pik1-83 allele did not enhance this defect but reduced somewhat the amount of cells having mislocalized ASH1 mRNA.

We also found that a temperature-sensitive allele of calmodulin (cmd1-239), which is known to affect actin-dependent endocytosis (33), led to the mislocalization of ASH1 mRNA to the mother cell in particular (Table IV and Fig. 1C). Therefore, a wide variety of mutants that play a role in endomembrane trafficking also appear to affect mRNA transport and localization in the daughter cell.

In Vivo Localization of ASH1 mRNA in Mutants of the Late Secretory Pathway—We verified the results obtained with the in situ hybridization approach by using live yeast cells expressing a viral MS2 protein-binding site fused to the 3'-untranslated region of ASH1. This mRNA transcript allows for binding of an MS2 coat protein-GFP fusion protein (MS2-GFP) and subsequent visualization of the mRNA particle when co-expressed (28). We found that MS2-GFP fluorescence localized exclusively to the daughter cells in wild-type yeast, whereas in myo4{Delta} cells MS2-GFP was prominent only in the mother cells (Fig. 2A), as expected.



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  		FIG. 2.
In vivo localization of ASH1 mRNA in mutants of the late secretory pathway. Yeast cells expressing an ASH1 gene encoding an MS2-binding site in its 3'-untranslated region and MS2-GFP from plasmids were grown to log phase and examined by fluorescence microscopy. A, in vivo localization of ASH1 mRNA in wild-type (WT), myo4{Delta}, and cdc42-6 cells. Both wild-type and myo4{Delta} cells were maintained at 26 °C, whereas a sample of the cdc42-6 cells were shifted to 33 °C for 1 h. Cells were then mounted in synthetic medium containing 0.5% agarose and visualized by confocal fluorescence microscopy. GFP indicates GFP fluorescence; GFP/Nom indicates the merge between GFP fluorescence and the cells visualized using Nomarski optics. B, in vivo localization of ASH1 mRNA in other mutants of the late secretory pathway. Both wild-type and secretory mutant cells were maintained at 26 °C or shifted to 37 °C for either 30 and 60 min and then visualized by fluorescence microscopy.

 
We next examined the localization of MS2-GFP in several of the secretory mutants found to affect ASH1 mRNA localization in situ as well as in other untested cell types. We found that MS2-GFP was localized to the mother cells in cdc42-6 cells at both permissive and restrictive temperatures (Fig. 2A). This verifies that the localization of the ASH1 mRNA particles is strongly altered in this mutant, as predicted from the in situ experiments (Fig. 1A and Table II). In other experiments, we found that the localization of the MS2-GFP particles was often partly affected even at permissive temperatures in yeast mutants of SNAREs (e.g. sec9-4), SNARE regulatory proteins (e.g. sec1-1), secretory GTPases (e.g. sec4-8), exocyst components (e.g. sec15-2), a vesicle transport myosin (e.g. myo2-66), and others (e.g. gga1{Delta} gga2{Delta}) (Fig. 2B).2 Under these conditions the MS2-GFP particles were often associated with the bud neck, instead of the bud tip, even though the cells appeared to be in early G2/M phase (Fig. 2B, see the right panels of 26 °C for specific examples). However, after the shift to restrictive temperatures, the particles became mislocalized prominently to the mother cells and were excluded from the daughter cells in many cases. This mislocalization was observed in the mutants shown in Fig. 2B, as well as in other mutants (e.g. sec9-7, arf1{Delta}, and sso2-1 cells) examined at restrictive temperatures.2 Moreover, the onset of MS2-GFP mislocalization was observed within 30 min (or less) after the temperature shift and remained that way for over 60 min. In contrast, the MS2-GFP particles in wild-type cells, although sometimes appearing more diffuse shortly after the temperature shift, were typically localized to the bud tip at 37 °C (Fig. 2A)2 at all times.

Secretory Mutants That Affect ASH1 mRNA Localization Strongly Affect the Integrity of the Actin Cytoskeleton—Previous studies (2, 8-12) have directly linked actin cytoskeleton regulation to the transport and localization of ASH1 mRNA. Because we have identified widely diverse mutants of the late secretory pathway that affect ASH1 mRNA localization, we examined whether there is a common mechanistic basis for their effects. The logical possibility was that all of these mutants also affect actin organization. To test this hypothesis, we examined the actin cytoskeleton in these mutants at permissive and restrictive temperatures using rhodamine-conjugated phalloidin and fluorescence microscopy. Indeed, we found a direct correlation between the disorganization of actin (as judged by the presence of actin patches in the mother cells and loss of actin cables in the bud) and the mislocalization of ASH1 mRNA in situ (see Table V for summarized results for actin organization and Fig. 3 for representative cells). Actin patches were very disorganized in cdc42-6, rho3-V51, ypt31{Delta} ypt32-A141D, and sec9-4 cells even at permissive temperatures. Notably, the extent of actin disorganization observed in these mutants at the permissive temperatures appeared to correlate with the extent of ASH1 mislocalization at the same temperatures, i.e. both actin and mRNA were highly mislocalized in cdc42-6, rho3-V51, and ypt31{Delta} ypt32-A141D cells, etc. At restrictive temperatures actin patches were completely disorganized and actin cables absent in all of the mutants examined, although no effect upon was observed in wild-type cells under the same conditions. As both the length and extent of the temperature shifts used in these experiments were basically identical to those used by other groups to examine secretory defects (i.e. invertase secretion or vesicle accumulation), we conclude that all the secretory mutations examined here disrupt the actin cytoskeleton to some degree. Thus, there appears to be a direct relationship between the integrity of the secretory pathway and the actin cytoskeleton (and not just vice versa). Moreover, this phenomenon appears to translate into defects in mRNA transport, as noted in this study.



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  		FIG. 3.
Actin deregulation is observed in mutants of the late secretory pathway. Yeast were grown to log phase on rich medium and either maintained at 26 °C (Permissive) or shifted to restrictive temperatures (Restrictive) prior to fixation and in situ labeling with rhodamine-conjugated phalloidin. Strains included pik1-83, sec1-1, sec4-8, sec9-4, sec15-2, sso2-1, and ypt31{Delta} ypt32-A141D (restrictive, 1 h at 37 °C); cdc42-6 (restrictive, 1 h at 33 °C); sro7{Delta} sro77{Delta} (restrictive, 1 h at 15 °C); rho3-V51 (restrictive, 1 h at 15 °C); and wild-type (WT) cells (1 h at 15, 18, or 37 °C).

 
The Onset of Actin and mRNA Transport Defects Is Rapid in Yeast Secretory Mutants—To test whether the onset of mRNA trafficking and actin defects are concurrent in these secretory mutants, we examined actin and ASH1 mRNA localization in time course experiments (Fig. 4). We examined this in sec15-2 cells, which show moderate defects in both actin organization and ASH1 mRNA localization at restrictive temperatures (Fig. 2B and Table III). sec15-2 cells expressing MS2-GFP and the MS2-binding site-tagged ASH1 mRNA were grown to log phase prior to shifting them to the appropriate elevated temperature for various lengths of time up to 60 min. ASH1 mRNA was localized in vivo (Fig. 4, A and B) and in situ in fixed cells2 by using fluorescence microscopy, whereas actin was examined by phalloidin labeling of the fixed cells (Fig. 4C). We found that both actin disorganization and MS2-GFP mislocalization occurred rapidly, with defects in actin being noticeable within 5 min, whereas defects in MS2-GFP localization were observed within approximately 15-20 min. We note that rapid disorganization of the actin cytoskeleton has been shown to occur in temperature-shifted wild-type cells (34) but becomes re-ordered within 30 min, unlike in the sec mutant strains shown (Fig. 4).2 Moreover, the slower rate of MS2-GFP mislocalization (vis à vis actin) is probably a measure of the time it takes for pre-localized mRNA particles to disperse from the bud tips following actin deregulation. Nonetheless, these results imply that both defects occur within the time period in which vesicular transport defects become evident. They also suggest that actin disorganization occurs prior to changes in the localization of anchored mRNA.



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  		FIG. 4.
mRNA and actin localization defects occur rapidly in late-acting secretory mutants. Yeasts expressing an ASH1 gene fragment encoding an MS2-binding site in its 3'-untranslated region and MS2-GFP from plasmids were grown to log phase on synthetic medium. Samples of cells were shifted to restrictive temperatures and either examined concurrently by fluorescence microscopy (GFP) as a function of time (0-50 min) or fixed, labeled with rhodamine-conjugated phalloidin, and examined in situ by fluorescence microscopy. A, GFP fluorescence in sec15-2 cells at the permissive temperature (26 °C). White arrowheads designate the MS2-GFP particles. Note their constraint to the bud tip throughout the experiment. B, GFP fluorescence in sec15-2 cells at the restrictive temperature (37 °C). Note the movement of the MS2-GFP particles away from the bud tip to the bud neck or into the mother cell within 20 min. The picture shown at 0 min is a compilation of several z series increments, whereas the pictures taken at other times are of a single z series section. C, phalloidin labeling of actin in sec15-2 cells at the restrictive temperature (37 °C). Note the rapid dispersal of actin organization within 5 min, and the appearance of disorganized actin patches within 30 min of the temperature shift. D, effect of cycloheximide on actin and ASH1 mRNA localization. Wild-type cells were incubated with (+) or without (-) cycloheximide (20 µg/ml) for 60 min, prior to the examination of ASH1 mRNA localization in vivo (MS2-GFP) by fluorescence microscopy or fixation and phalloidin labeling in situ (Actin). E, in vivo localization of ASH1 mRNA in a mutant of the early secretory pathway. sec22-2 cells expressing the MS2-binding site-tagged ASH1 and MS2-GFP from plasmids were grown to log phase on synthetic medium and either maintained at 26 °C or shifted to the restrictive temperature (37 °C) for 1 h. Cells were examined for ASH1 mRNA localization in vivo (MS2-GFP) by fluorescence microscopy or for actin after fixation and phalloidin labeling in situ (Actin).

 
We next examined whether changes in protein synthesis, which may occur in sec mutants after a certain time, have an effect upon actin organization and ASH1 localization (Fig. 4D). Wild-type cells expressing MS2-GFP and the binding site-tagged ASH1 gene were treated with or without cycloheximide (20 µg/ml) prior to the examination of ASH1 mRNA localization in vivo and actin staining in situ. No changes in either ASH1 mRNA localization or actin labeling were observed for up to 60 min of treatment. This result was not unexpected, however, because cycloheximide treatment per se does not block the exocytosis of accumulated vesicles or endocytosis (35, 36). This suggests then that ASH1 mRNA transport, actin organization, and the results observed in this study are probably independent of alterations in protein synthesis within the time period examined. Finally, we tested whether mutations in the early secretory pathway also affect actin organization and ASH1 mRNA localization (Fig. 4E). We examined ASH1 mRNA localization in vivo and actin staining in situ in sec22-2 cells that were maintained at 26 °C or shifted to 37 °C for 30-60 min. We found that both ASH1 mRNA and actin were mislocalized at the elevated temperature. Because transport along the late secretory pathway (i.e. post-Golgi) is dependent upon the integrity of the early pathway (i.e. endoplasmic reticulum-Golgi), but not vice versa, this result was also expected.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Polarity in yeast cells is achieved by the polarized organization of actin in a manner that is dependent upon the spatial regulation of exocytosis. This predicts that actin organization and exocytosis are coupled events, an idea supported by several recent studies (37, 38). At the same time, mRNA transport and localization are also dependent upon the integrity of the actin cytoskeleton in yeast (2, 4, 8-12). Thus, we predict that proteins directly involved in actin-dependent exocytosis might also influence actin-dependent mRNA transport.

Here, we demonstrate that a wide variety of late-acting secretory mutants have defects in mRNA transport and localization to the daughter cells. This is evidenced by performing in situ hybridization with probes directed against ASH1 mRNA, as well as in in vivo fluorescence studies using an ASH1 mRNA containing an MS2-binding site (28, 39). The results imply that proteins involved in Golgi export (i.e. Pik1 and Ypt31/32), secretory vesicle tethering (i.e. Sec4, Sec10, and Sec15), SNARE regulation (i.e. Sec1 and Sro7), and the SNAREs themselves (i.e. Sec9 and Sso1/2) all affect ASH1 mRNA localization (Tables II, III, IV; see Figs. 1 and 2 for specific examples). In addition, alleles of other small GTPases proposed to affect exocytosis (i.e. cdc42-6 and rho3-V51) (16, 17) also had strong effects upon ASH1 mRNA distribution and resulted in a high level of asymmetric mRNA distribution to the mother cell (Table II).

Because these and several other mutants tested (i.e. ras2{Delta} and cmd1-239), along with the known cytoskeleton mutants (i.e. tpm1{Delta}, tpm1-2 tpm2{Delta}, myo4{Delta}, and myo2-66) (Table IV), all show defects in ASH1 mRNA localization, we assumed that some common attribute must connect the functioning of these diverse proteins. We hypothesized that this was likely to reside at the level of actin cytoskeleton regulation, and therefore, we tested the various mutants for defects in actin organization (Fig. 3). Indeed, we found a strong correlation between the loss of actin polarity (Figs. 3 and 4) and the mislocalization of ASH1 mRNA in the secretory mutants (Figs. 1, 2, and 4). Thus, secretory mutations that alter the actin cytoskeleton also affect ASH1 mRNA transport and localization, as well as exocytosis. Perhaps not surprisingly, the two small GTPase mutants (i.e. rho3-V51 and cdc42-6) that affected ASH1 mRNA localization, but had been proposed to affect vesicle transport and not the actin cytoskeleton per se (16, 17), also had defects in actin polarity even at permissive temperatures. Thus, the secretory defects observed in these mutants (16, 17) are likely to result from defects in actin-dependent vesicle transport and not, necessarily, from defects in vesicle biogenesis or fusion.

A number of secretory mutants have been shown individually to affect actin polarity, including sec1-1 (34), sec4-8 (40), and pik1-83 (21), as demonstrated here (Figs. 3 and 4), as well as sec3 (41) and sec6-4 (34). However, the specific contribution of these proteins to actin organization (and subsequent mRNA transport) has not been fully appreciated. That all secretory mutants affect both the cytoskeleton and mRNA transport raises the issue of coupling between active secretion, actin regulation, and mRNA localization. Conventional wisdom would suggest that as exocytosis becomes blocked (i.e. at restrictive temperatures in a temperature-sensitive secretion mutant), there is either a concomitant or subsequent loss in actin regulation as well. This could result, for example, from the loss of polarity factors (i.e. Cdc42) at the site of secretion (38, 42) if their delivery is dependent upon either mRNA transport, vesicular transport, or both. Most interestingly, recent work (38) demonstrated that expression of an activated form of Cdc42 in nonpolarized G1-arrested cells results in random polarization, rather than the spatially regulated budding pattern typically observed. The localization and enrichment of Cdc42 at the site of exocytosis, either due to its delivery by secretory vesicles or local translation of CDC42 mRNA, may then establish cell polarity and reinforce it via actin regulation. Thus, regulatory elements of the cytoskeleton (i.e. Cdc42 and Rho3) that are shared with the actin-dependent secretory pathway are also candidates for control of the actin-dependent mRNA localization pathway.

Alternatively, because such a wide variety of secretory mutations leads to actin depolarization, it is possible that vesicular transport factors, be they cytoskeletal components (e.g. Myo2), SNARE regulators (e.g. Sec1), or components of the docking and fusion machinery (e.g. Sec4, Exocyst, and SNAREs, etc.), all impinge somehow upon functioning of the Formins and Arp2/3 complex and their inherent ability to polymerize actin and drive organelle movement (4, 43, 44). Thus, onset of the secretory block may couple directly with the deregulation of actin and lead to a subsequent loss in mRNA transport and/or localization. Most interestingly, the rapidity in which defects in actin are observed in temperature-shifted secretory mutants (Fig. 4) suggests that there may be an explicit signal sent to the actin regulatory machinery that is either generated or blocked upon the cessation of vesicle transport, docking, or fusion. Although the nature of this signal is not yet known, presumably it should transduce the occurrence of fusion events at the site of exocytosis directly to the actin cytoskeleton, perhaps via Cdc42 and/or Rho1. A role for phosphoinositide signaling, which generates transient membrane-localized signals and modulates the actin cytoskeleton, in this potential "exocytic signal" should be explored.

Another question that arises is how much of the secretory block is reinforced by the subsequent defects in mRNA transport and localization? Most interestingly, initial work in our laboratory strongly suggests that mRNAs encoding a number of secretory components are asymmetrically distributed to the daughter cell. Therefore, specific defects in mRNA transport could result in protein depletion at the site of exocytosis and contribute to the loss in exocytic capacity3 and perhaps polarity. That said, it is apparent that whereas vesicle transport and mRNA localization to the bud are largely coupled, mutations that disrupt mRNA transport (i.e. myo4{Delta}, tpm1{Delta}, etc.) do not completely block exocytosis and result only in partial secretion defects or the accumulation of secretory vesicles (6, 45). Thus, mRNA localization may principally affect the establishment of cell polarity and the distribution of inheritance factors but not necessarily overall cell growth and viability. An example of this is the nonpolarized type of exocytosis that occurs in tpm1{Delta} and tpm1-2 tpm2{Delta} mutant cells (4, 6). Thus, cells retain the ability to maintain active sites of exocytosis even in the absence of proper mRNA transport. This suggests that additional means exist to ensure the delivery of secretory proteins to the sites of secretion. Nonetheless, the connection between these three separate, but perhaps mutually interdependent processes (e.g. actin organization, mRNA transport, and exocytosis) (see Fig. 5), should be carefully considered when evaluating the contribution of proteins to endomembrane trafficking in eukaryotic cells.



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  		FIG. 5.
A model for the interdependence of the vesicle and mRNA transport pathways and the actin cytoskeleton. The integrity of the actin cytoskeleton is known to be essential for both vesicle and mRNA transport to the growing bud in yeast. Here we have demonstrated that mutations in genes encoding proteins specifically involved in transport along the late secretory pathway influence the integrity of the cytoskeleton as well as mRNA transport. Thus, these three processes are interconnected and presumably are interdependent. Open questions (?) remain regarding the connection between mRNA transport and its ability to influence both vesicle transport and organization of the actin cytoskeleton. If polarity factors (i.e. Cdc24/Cdc42 and Rom2/Rho1) localize to the site(s) of secretion as a result of local protein translation, then mRNA transport can be expected to play an important role in the maintenance of polarized growth.

 

   FOOTNOTES
 
* This work was supported in part by a grant from the Minerva Foundation, Germany (to J. E. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by the Forchheimer Center for Molecular Genetics and by an Ann Abrams Stone post-doctoral fellowship from the Feinberg Graduate School. Back

§ Holds the Henry Kaplan Chair in Cancer Research. To whom correspondence should be addressed. Tel.: 972-8-9342106; Fax: 972-8-9344108; E-mail: jeffrey.gerst{at}weizmann.ac.il.

1 The abbreviations used are: SNARE, soluble NSF attachment protein receptor; GFP, green fluorescent protein; PBS, phosphate-buffered saline; FISH, fluorescence in situ hybridization; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back

2 S. Aronov and J. E. Gerst, unpublished observations. Back

3 S. Aronov and J. E. Gerst, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Kerry Bloom (University of North Carolina, Chapel Hill, NC) for generously supplying plasmids. We also thank Patrick Brennwald (University of North Carolina), Tony Bretscher (Cornell University), Scott Emr (University of California, San Diego), Eitan Gross (Weizmann Institute, Israel), Tim Huffaker (Cornell University), Peter Novick (Yale University), Pak Poon (Dalhousie University), Hugh Pelham (MRC, Cambridge, UK), Howard Riezman (University of Geneva, Switzerland), and Hans Ronne (Swedish University of Agricultural Science, Uppsala, Sweden) for yeast strains.



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