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Two new Ypt GTPases are required for exit from the yeast trans-Golgi compartment.

Jedd G, Mulholland J, Segev N - J. Cell Biol. (1997)

Bottom Line: These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions.The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells.Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the trans-Golgi compartment, between the reactions regulated by Ypt1p and Sec4p.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Physiological Sciences, The University of Chicago, Illinois 60637, USA.

ABSTRACT
Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the trans-Golgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.

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ypt31-Δ/ypt32-A141D (NSY313) and sec4-G147D (PNY404) mutant cells both show defects in the trans-Golgi processing and  transport of α-factor at the nonpermissive temperature. (A) The indicated strains (wild-type is NSY128) were labeled for 7 min at 26°C,  shifted to 37°C with prewarmed media, and chased for the indicated times. Cells were separated from medium by centrifugation and  processed for immunoprecipitation with anti–α-factor antibodies. (B) Alternatively, cells were preshifted to 37°C for 2 min with prewarmed medium, labeled for 7 min and chased for the indicated times. Only mature peptide is shown, as higher molecular weight forms  of α-factor behave similarly to those shown in A, except that both mutants secreted proportionally less high molecular weight α-factor  after the preshift, and all strains (including wild-type) show translocation and processing defects. Positions of ER (core), cis-Golgi (α16), medial-Golgi (α1-3), and trans-Golgi (mαf) forms are noted in the left margin. Arrows in the right margin indicate secreted α-factor  that was not fully processed in the trans-Golgi.
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Figure 5: ypt31-Δ/ypt32-A141D (NSY313) and sec4-G147D (PNY404) mutant cells both show defects in the trans-Golgi processing and transport of α-factor at the nonpermissive temperature. (A) The indicated strains (wild-type is NSY128) were labeled for 7 min at 26°C, shifted to 37°C with prewarmed media, and chased for the indicated times. Cells were separated from medium by centrifugation and processed for immunoprecipitation with anti–α-factor antibodies. (B) Alternatively, cells were preshifted to 37°C for 2 min with prewarmed medium, labeled for 7 min and chased for the indicated times. Only mature peptide is shown, as higher molecular weight forms of α-factor behave similarly to those shown in A, except that both mutants secreted proportionally less high molecular weight α-factor after the preshift, and all strains (including wild-type) show translocation and processing defects. Positions of ER (core), cis-Golgi (α16), medial-Golgi (α1-3), and trans-Golgi (mαf) forms are noted in the left margin. Arrows in the right margin indicate secreted α-factor that was not fully processed in the trans-Golgi.

Mentions: To distinguish between a block in the medial- or transGolgi compartments, which contain α-1,3-mannosyltransferase and Kex2 protease, respectively, we followed the processing of α-factor. This protein contains recognition sites for the Kex2 protease. Wild-type, ypt31-Δ/ypt32-A141D, and sec4-G147D mutant cells were labeled at the permissive temperature (26°C) for 7 min, shifted to the nonpermissive temperature (37°C), and chased for 10 or 30 min. Cells were separated from the medium, and α-factor was immunoprecipitated and analyzed by gel electrophoresis. In wild-type and mutant cells, the various secretory compartments were loaded with the different forms of α-factor during the pulse, as seen by the presence of core, α-1,6modified, α-1,3-modified, and mature forms (Fig. 5, chase time 0′). In wild-type cells, even after a short chase, all of these forms were converted to the mature form. The limitation of using α-factor as a marker is that the mature form cannot be quantitatively detected, probably because of degradation, and the analysis of the processing is thus restricted to determining the disappearance of the intermediate forms. sec4-G147D mutant cells exhibited a defect in secretion of mature α-factor consistent with a defect in the fusion of post-Golgi vesicles with the plasma membrane (Fig. 5 A, mαf). Like sec4-G147D mutant cells, ypt31-Δ/ ypt32-A141D mutant cells were defective in the secretion of mature α-factor peptide but were also partially defective at earlier stages of the secretory pathway. In addition, both mutants secreted some α-factor that had not received proteolytic processing, indicating an additional defect in the function of the trans-Golgi (Fig. 5 A, arrows). However, this trans-Golgi processing defect is probably minor and secondary, since the immature forms accumulated after the chase in both mutant strains represent only a minor fraction of the α-factor labeled during the pulse (time 0). Because a significant amount of α-factor is secreted under the conditions of this experiment, we examined the secretion of α-factor after a 2-min preshift to the nonpermissive temperature. Under these conditions, less α-factor is secreted to the medium in both the sec4 and ypt31/32 mutants (Fig. 5 B), although the ypt31/32 mutant is still slightly leaky. Together, the results suggest that ypt31/32 mutant cells, like sec4 mutant cells, are defective in processes that occur in the exit from the trans-Golgi, or within this compartment.


Two new Ypt GTPases are required for exit from the yeast trans-Golgi compartment.

Jedd G, Mulholland J, Segev N - J. Cell Biol. (1997)

ypt31-Δ/ypt32-A141D (NSY313) and sec4-G147D (PNY404) mutant cells both show defects in the trans-Golgi processing and  transport of α-factor at the nonpermissive temperature. (A) The indicated strains (wild-type is NSY128) were labeled for 7 min at 26°C,  shifted to 37°C with prewarmed media, and chased for the indicated times. Cells were separated from medium by centrifugation and  processed for immunoprecipitation with anti–α-factor antibodies. (B) Alternatively, cells were preshifted to 37°C for 2 min with prewarmed medium, labeled for 7 min and chased for the indicated times. Only mature peptide is shown, as higher molecular weight forms  of α-factor behave similarly to those shown in A, except that both mutants secreted proportionally less high molecular weight α-factor  after the preshift, and all strains (including wild-type) show translocation and processing defects. Positions of ER (core), cis-Golgi (α16), medial-Golgi (α1-3), and trans-Golgi (mαf) forms are noted in the left margin. Arrows in the right margin indicate secreted α-factor  that was not fully processed in the trans-Golgi.
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Related In: Results  -  Collection

Show All Figures
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Figure 5: ypt31-Δ/ypt32-A141D (NSY313) and sec4-G147D (PNY404) mutant cells both show defects in the trans-Golgi processing and transport of α-factor at the nonpermissive temperature. (A) The indicated strains (wild-type is NSY128) were labeled for 7 min at 26°C, shifted to 37°C with prewarmed media, and chased for the indicated times. Cells were separated from medium by centrifugation and processed for immunoprecipitation with anti–α-factor antibodies. (B) Alternatively, cells were preshifted to 37°C for 2 min with prewarmed medium, labeled for 7 min and chased for the indicated times. Only mature peptide is shown, as higher molecular weight forms of α-factor behave similarly to those shown in A, except that both mutants secreted proportionally less high molecular weight α-factor after the preshift, and all strains (including wild-type) show translocation and processing defects. Positions of ER (core), cis-Golgi (α16), medial-Golgi (α1-3), and trans-Golgi (mαf) forms are noted in the left margin. Arrows in the right margin indicate secreted α-factor that was not fully processed in the trans-Golgi.
Mentions: To distinguish between a block in the medial- or transGolgi compartments, which contain α-1,3-mannosyltransferase and Kex2 protease, respectively, we followed the processing of α-factor. This protein contains recognition sites for the Kex2 protease. Wild-type, ypt31-Δ/ypt32-A141D, and sec4-G147D mutant cells were labeled at the permissive temperature (26°C) for 7 min, shifted to the nonpermissive temperature (37°C), and chased for 10 or 30 min. Cells were separated from the medium, and α-factor was immunoprecipitated and analyzed by gel electrophoresis. In wild-type and mutant cells, the various secretory compartments were loaded with the different forms of α-factor during the pulse, as seen by the presence of core, α-1,6modified, α-1,3-modified, and mature forms (Fig. 5, chase time 0′). In wild-type cells, even after a short chase, all of these forms were converted to the mature form. The limitation of using α-factor as a marker is that the mature form cannot be quantitatively detected, probably because of degradation, and the analysis of the processing is thus restricted to determining the disappearance of the intermediate forms. sec4-G147D mutant cells exhibited a defect in secretion of mature α-factor consistent with a defect in the fusion of post-Golgi vesicles with the plasma membrane (Fig. 5 A, mαf). Like sec4-G147D mutant cells, ypt31-Δ/ ypt32-A141D mutant cells were defective in the secretion of mature α-factor peptide but were also partially defective at earlier stages of the secretory pathway. In addition, both mutants secreted some α-factor that had not received proteolytic processing, indicating an additional defect in the function of the trans-Golgi (Fig. 5 A, arrows). However, this trans-Golgi processing defect is probably minor and secondary, since the immature forms accumulated after the chase in both mutant strains represent only a minor fraction of the α-factor labeled during the pulse (time 0). Because a significant amount of α-factor is secreted under the conditions of this experiment, we examined the secretion of α-factor after a 2-min preshift to the nonpermissive temperature. Under these conditions, less α-factor is secreted to the medium in both the sec4 and ypt31/32 mutants (Fig. 5 B), although the ypt31/32 mutant is still slightly leaky. Together, the results suggest that ypt31/32 mutant cells, like sec4 mutant cells, are defective in processes that occur in the exit from the trans-Golgi, or within this compartment.

Bottom Line: These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions.The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells.Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the trans-Golgi compartment, between the reactions regulated by Ypt1p and Sec4p.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Physiological Sciences, The University of Chicago, Illinois 60637, USA.

ABSTRACT
Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the trans-Golgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.

Show MeSH
Related in: MedlinePlus