Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex - PubMed (original) (raw)

Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex

Olivier Roumanie et al. J Cell Biol. 2005.

Abstract

Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3-Exo70 and Rho1-Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Distribution of the polarity and secretion markers Cdc42, Myo2, and Sec4 is normal in the rho3-V51 secretory mutant at both permissive and nonpermissive temperatures. (A) Wild-type (WT) and mutant (rho3-V51) cells were grown at 25°C or shifted to 14°C for 5 h before fixation and then processed for fluorescence microscopy. Visualization of the polarity establishment protein Cdc42, the Rab GTPase Sec4, and the vesicle-transport motor Myo2 was performed using specific purified antibodies. Bar, 2 μm. (B) Quantitation of polarized markers in wild-type and rho3-V51 cells. Cells were processed as described in A and scored for the polarized localization of Cdc42, Sec4, and Myo2 at the emerging bud sites, bud tips, or mother-daughter neck regions. A minimum of 200 cells were counted for each experiment.

Figure 2.

Figure 2.

Exocyst subunits Sec3, Sec8, and Exo70 are polarized in the rho3-V51 secretory mutant at permissive and nonpermissive temperatures. (A) Plasmids containing functional, COOH-terminal–tagged GFP versions of Sec3, Sec8, or Exo70 were introduced into wild-type and rho3-V51 strains. Transformed cells were grown at 25°C or shifted to 14°C for 5 h and immediately fixed and processed for fluorescence microscopy. DIC images of the same cells are shown. Bar, 2 μm. (B) Quantitation of Sec3-GFP, Sec8-GFP, and Exo70-GFP polarized localization in wild-type and rho3-V51 cells. Cells processed as described in A were scored for the presence of the exocyst components at the emerging bud sites, bud tips, or bud necks. Approximately 50 cells were analyzed under each condition. (C) Invertase and Bgl2 secretion assays on GFP-tagged wild-type and rho3-V51 strains processed as described in A were performed as described previously (Adamo et al., 1999). The secretion defect associated with each strain is expressed as the percentage of total (internal + external) invertase or Bgl2 found internally. Results shown represent the average of three experiments. Error bars represent SD.

Figure 3.

Figure 3.

Testing the redundancy model for Rho function in the polarization of secretory and polarity markers. (A) Redundancy model for Rho3 and Rho1/Cdc42 pathways in polarizing the exocyst. The Exo70 and Sec3 exocyst subunits interact with distinct Rho GTPases, and these interactions have been proposed to localize the exocyst complex to help promote secretion and polarity. (B) A sec3 mutant strain lacking the NH2-terminal RBD (aa 3–320) of Sec3 was placed as the only copy of Sec3 in the cell on high copy expression (see Materials and methods). Correct integration of the construct was checked by Western blot analysis of whole cell lysates from wild-type and _sec3-Δ_N strains using anti-Sec3 antibodies directed against the middle region of the protein. Blotting with anti-Exo70 antibodies was used as loading control. (C) Polarization of distinct secretion markers in the _sec3-Δ_N mutant. Wild-type and _sec3-Δ_N cells were grown at 25°C, fixed, and processed for immunofluorescence. Specific purified antibodies directed against the Sec3 and Sec15 exocyst subunits, the polarity protein Cdc42, or the secretion regulator Sec4 were used. Bar, 2 μm. (D) Quantitation of Cdc42, Sec4, and Sec15 polarization in the _sec3-Δ_N mutant at 25°C. The polarized distribution of the three proteins was scored as described in Fig. 1. (E) Measurement of the relative fluorescence associated with the Cdc42, Sec4, and Sec15 markers in small budded cells at 25°C. Proteins were detected by immunofluorescence microscopy in wild-type and _sec3-Δ_N cells, and the fluorescence intensity of the polarized spots was measured in small budded cells using Metamorph software. Results for each marker are reported as an intensity of fluorescence relative to the fluorescence observed in the wild-type strain for the same marker. Error bars represent SD.

Figure 4.

Figure 4.

_rho3-V5_1 and sec3- ΔN mutations do not demonstrate any synthetic effects on growth and secretion. (A) Analysis of the growth phenotypes of the _rho3-V51,sec3-Δ_N double mutant. The sec3-2 and sec4-P48 strains were used as growth controls. Plates at 37 and 25°C were incubated for 2–3 d or at 14°C for 6 d. (B) Analysis of Bgl2 protein secretion in wild-type, _sec3-Δ_N, rho3-V51, and _rho3-V51,sec3-Δ_N strains. Cells were grown at 25°C or shifted to 14°C for 5 h before analyzing the distribution of the secreted protein Bgl2 as described previously (Adamo et al.,1999). Quantitation of the bands was performed using ImageQuant software. The percentage of the total Bgl2 that is found internally in different strains is depicted. The data represent the average of three independent experiments. Error bars represent SD.

Figure 5.

Figure 5.

_rho3-V51,sec3-Δ_N cells show no defect in the polarization of polarity or secretory markers. (A) The _rho3-V51,sec3-Δ_N cells were grown at 25°C and shifted to 14°C for 5 h before being analyzed by fluorescence microscopy. Purified antibodies were used to visualize polarity/secretion markers (Cdc42, Sec4, and Myo2) and exocyst subunits (Sec3 and Sec15). Bar, 2 μm. (B) Quantitation of polarized markers at 14°C in the indicated wild-type or mutant strains. Cells were scored for the polarized localization of Cdc42, Sec4, Myo2, and Sec15 as described in Fig. 1. (C) Measurement of relative fluorescence associated with the polarity/secretion markers in small budded cells at 14°C. Polarized staining was analyzed as described in Fig. 3. Error bars represent SD. (D) The plasmids carrying the GFP-tagged exocyst subunits Sec8 or Exo70 were transformed into the _rho3-V51,sec3-Δ_N double mutant strain. The cells were grown at 25°C, shifted to 14°C for 5 h, fixed, and observed by fluorescence and DIC microscopy. Bar, 2 μm. (E) Quantitation of Sec8-GFP and Exo70-GFP polarized localization in wild-type and mutant strains at 14°C. Under each condition, ∼50 cells were analyzed and scored for the presence of the exocyst components at the emerging bud sites, bud tips or bud neck.

Figure 6.

Figure 6.

Sec8-GFP photobleaching and FRAP measurements in rho3-V51,sec3- Δ_N_ cells show wild-type recovery times. (A) Representative example of Sec8-GFP photobleaching experiment. Arrow denotes photobleached area. Prebleach, t = −3 s; photobleaching, t = 0 s; maximum recovery, t = 105 s. Bar, 2 μm. (B) Measured fluorescence recovery of Sec8-GFP (black squares) compared with single exponential fit of recovery (gray triangles). Fluorescence intensity is recorded in arbitrary units (A.U.). See Materials and methods for details of analysis. (C) Average half-recovery times measured for Sec8-GFP. (D) Average percent recovery values for Sec8-GFP. Error bars represent SD.

Figure 7.

Figure 7.

A functional secretory pathway is required to polarize the exocyst subunits Sec3 and Sec15 and the myosin Myo2. Sec1-1, sec6-4, and myo2-66 mutants, which are blocked at a late stage in transport, and the early secretion mutant sec22-3 and wild-type cells were grown at 25°C and shifted to 37°C for 1 h, fixed, and processed for fluorescence microscopy. Purified antibodies directed against the Rab protein Sec4, the type V myosin Myo2, and the Sec3 and Sec15 exocyst subunits were used for immunostaining. Bar, 2 μm.

Figure 8.

Figure 8.

The polarized localization of Sec3, Sec15, and Sec4 is dependent on actin cables. (A) Polarization of Sec3 and Sec4 in _tpm1-2, tpm2_Δ homozygous diploids and control _tpm2_Δ diploid cells (with functional TPM1; both gifts of A. Bretscher, Cornell University, Ithaca, NY) was examined by double-label immunofluorescence after temperature shifts to 34°C for the indicated times. Bar, 1 μm. (B) Shift to the restrictive temperature rapidly disrupts the polarization of Sec3, Sec15, and Sec4 in tropomyosin mutant strains. Cells were shifted for the indicated times and immediately fixed. Small budded cells were scored for the polarization of Sec3, Sec15, and Sec4 after immunofluorescent staining. Small budded cells were defined as cells whose buds were less than half the size of the mother cell. 200–300 cells were scored per data point.

Figure 9.

Figure 9.

GTP hydrolysis by Rho3 and Cdc42 is not essential for their secretory function. (A, i) sec4-P48 cs phenotype is efficiently suppressed by the activated RHO3 Q74L allele. Wild-type and mutant sec4-P48 strains were transformed with single-copy (CEN) plasmids with the indicated gene, picked into microtiter wells, and transferred onto SC-ura plates at 25 and 14°C. Similar results were obtained on YPD medium. (ii) GTP hydrolysis by Rho3 is not necessary for its function in secretion. Centromeric plasmids containing the wild-type or activated alleles of RHO3 were introduced into the secretion-defective mutant rho3-V51 and wild-type strain. Suppression of cs phenotype was assayed as described for A (i). (iii) Constitutively activated Rho3 is a strong suppressor of the cdc42-6 secretion mutant. RHO3 and RHO3 Q74L centromeric plasmids were transformed into wild-type and cdc42-6 cells. Suppression of the growth defect was tested on YPD media at the restrictive temperatures of 32 and 37°C. (iv) Complementation of the rho3 deletion by activated RHO3 Q74L. A _rho3_Δ strain containing CEN/URA3/RHO3 plasmid was used in a plasmid shuffle complementation assay. This strain was transformed with an empty CEN/HIS3 plasmid or an identical plasmid containing wild-type RHO3, activated RHO3 Q74L, or the GDP-blocked mutant RHO3 T30N. Transformants were tested for growth on control (SD) plates or 5-FOA plates, which select against the CEN/URA3/RHO3 plasmid, to monitor growth of each strain with the CEN/HIS3 plasmid as the sole source of RHO3 in the cell. Plates were grown for 3 d at 30°C. (B) GTP hydrolysis by Cdc42 is not necessary for suppression of the cdc42-6 secretion defect. GAL1 EG43 plasmid carrying the wild-type or activated (Q61L) allele of CDC42 was integrated into the URA3 locus of the wild-type, cdc42-6, cdc42-17, and cdc42-27 strains. Empty GAL1 EG43 vector was used as a growth control. Colonies from each transformation were picked into microtiter wells and transferred onto YP plates containing either 2% glucose or 2% galactose to repress or induce the EG43 crippled GAL1 promoter, respectively. The cells were then incubated at the permissive temperature (25°C) or at the nonpermissive temperatures (32°C and 37°C) for 2–3 d.

Figure 10.

Figure 10.

Models for the spatial regulation of exocytosis by Rho GTPases. (A) Recruitment model for regulation of exocytosis. A polarized patch of GTP-bound Rho protein would recruit specific components of the exocytic machinery, including the exocyst complex, to a specific site on the plasma membrane. Once polarized, the exocyst complex would act as a spatial landmark for exocytosis, restricting the docking and fusion of post-Golgi vesicles at this site. (B) Allostery model for regulation of exocytosis. A polarized patch of activated Rho GTPase would act directly as a spatial landmark for exocytosis by locally activating the late secretory machinery, including the exocyst complex. This locally activated machinery would increase the likelihood of productive docking and fusion at this site. Many components of the exocytic machinery, including Rho GTPases and exocyst subunits, have been shown to be associated with secretory vesicles. Therefore, increased exocytosis at sites of allosteric activation would lead to both reinforcement of the activation signal as well as polarization of the exocytic machinery.

Similar articles

Cited by

References

    1. Abe, M., H. Qadota, A. Hirata, and Y. Ohya. 2003. Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae. J. Cell Biol. 162:85–97. - PMC - PubMed
    1. Adamo, J.E., G. Rossi, and P. Brennwald. 1999. The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity. Mol. Biol. Cell. 10:4121–4133. - PMC - PubMed
    1. Adamo, J.E., J.J. Moskow, A.S. Gladfelter, D. Viterbo, D.J. Lew, and P.J. Brennwald. 2001. Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud. J. Cell Biol. 155:581–592. - PMC - PubMed
    1. Ayscough, K.R., J. Stryker, N. Pokala, M. Sanders, P. Crews, and D.G. Drubin. 1997. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J. Cell Biol. 137:399–416. - PMC - PubMed
    1. Boyd, C., T. Hughes, M. Pypaert, and P. Novick. 2004. Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J. Cell Biol. 167:889–901. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources