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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 mutant cells do not exhibit a major defect in targeting of CPY to the vacuole at the nonpermissive temperature. Extracts from the 37°C samples shown in Fig. 4 were subjected to precipitation with anti-CPY antibodies and resolved on 8%  SDS–polyacrylamide gels. Positions of the ER (p1), Golgi (p2), and vacuolar (m) forms of CPY are indicated in the left margin.
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Figure 6: ypt31-Δ/ypt32-A141D mutant cells do not exhibit a major defect in targeting of CPY to the vacuole at the nonpermissive temperature. Extracts from the 37°C samples shown in Fig. 4 were subjected to precipitation with anti-CPY antibodies and resolved on 8% SDS–polyacrylamide gels. Positions of the ER (p1), Golgi (p2), and vacuolar (m) forms of CPY are indicated in the left margin.

Mentions: We wished to determine whether transport to the vacuole is affected by the ypt31/32 mutations. Proteins destined for the vacuole are sorted from secreted proteins at the trans-Golgi compartment (Graham and Emr, 1991). Thus, CPY processing is dependent on the function of SEC genes that function at all steps between ER and the transGolgi, and on VPS genes that regulate sorting at the transGolgi, but not on SEC genes that function at the post- trans-Golgi step of the exocytic pathway (Stevens et al., 1982). To examine CPY processing, the secretory compartments were loaded during a pulse at the permissive temperature with the various labeled forms of CPY (the p1 form in the ER, p2 form in the Golgi, and mature form in the vacuole) and then shifted to 37°C for the chase. In wild-type cells, all of the CPY was converted to the mature form after 30 min of chase. ypt31/32 mutant cells sorted most of their CPY to the vacuole as seen by the accumulation of the mature form (80% when compared to wildtype). However, CPY processing was somewhat slower in mutant cells than in wild-type cells: The ER form (p1) was still present after 10 min chase, and some CPY remained as the p2 form after 30 min of chase at the nonpermissive temperature in mutant, but not in wild-type cells (Fig. 6). Mutations in VPS genes result in secretion of the p2 form of CPY (Banta et al., 1988). Since no CPY was secreted by ypt31/32 mutant cells at the nonpermissive temperature, Ypt31/32 GTPases do not seem to function in the sorting of vacuolar proteins (Fig. 6). These results suggest that there is no major defect in the processing of vacuolar CPY in ypt31/32 mutant cells. The defect revealed by the accumulation of the p2 form might reflect a secondary defect at the trans-Golgi compartment after a 30-min incubation at the nonpermissive temperature, or a decrease in the rate of CPY processing through the whole pathway in mutant cells. Alternatively, Ypt31/32 GTPases might have a role in more than one step between the medial-Golgi and the plasma membrane.


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 mutant cells do not exhibit a major defect in targeting of CPY to the vacuole at the nonpermissive temperature. Extracts from the 37°C samples shown in Fig. 4 were subjected to precipitation with anti-CPY antibodies and resolved on 8%  SDS–polyacrylamide gels. Positions of the ER (p1), Golgi (p2), and vacuolar (m) forms of CPY are indicated in the left margin.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139891&req=5

Figure 6: ypt31-Δ/ypt32-A141D mutant cells do not exhibit a major defect in targeting of CPY to the vacuole at the nonpermissive temperature. Extracts from the 37°C samples shown in Fig. 4 were subjected to precipitation with anti-CPY antibodies and resolved on 8% SDS–polyacrylamide gels. Positions of the ER (p1), Golgi (p2), and vacuolar (m) forms of CPY are indicated in the left margin.
Mentions: We wished to determine whether transport to the vacuole is affected by the ypt31/32 mutations. Proteins destined for the vacuole are sorted from secreted proteins at the trans-Golgi compartment (Graham and Emr, 1991). Thus, CPY processing is dependent on the function of SEC genes that function at all steps between ER and the transGolgi, and on VPS genes that regulate sorting at the transGolgi, but not on SEC genes that function at the post- trans-Golgi step of the exocytic pathway (Stevens et al., 1982). To examine CPY processing, the secretory compartments were loaded during a pulse at the permissive temperature with the various labeled forms of CPY (the p1 form in the ER, p2 form in the Golgi, and mature form in the vacuole) and then shifted to 37°C for the chase. In wild-type cells, all of the CPY was converted to the mature form after 30 min of chase. ypt31/32 mutant cells sorted most of their CPY to the vacuole as seen by the accumulation of the mature form (80% when compared to wildtype). However, CPY processing was somewhat slower in mutant cells than in wild-type cells: The ER form (p1) was still present after 10 min chase, and some CPY remained as the p2 form after 30 min of chase at the nonpermissive temperature in mutant, but not in wild-type cells (Fig. 6). Mutations in VPS genes result in secretion of the p2 form of CPY (Banta et al., 1988). Since no CPY was secreted by ypt31/32 mutant cells at the nonpermissive temperature, Ypt31/32 GTPases do not seem to function in the sorting of vacuolar proteins (Fig. 6). These results suggest that there is no major defect in the processing of vacuolar CPY in ypt31/32 mutant cells. The defect revealed by the accumulation of the p2 form might reflect a secondary defect at the trans-Golgi compartment after a 30-min incubation at the nonpermissive temperature, or a decrease in the rate of CPY processing through the whole pathway in mutant cells. Alternatively, Ypt31/32 GTPases might have a role in more than one step between the medial-Golgi and the plasma membrane.

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