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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 exhibit a tight conditional secretory block of invertase transport downstream of  the medial-Golgi. Wild-type  (NSY349, left) and mutant  (NSY348, right) cells were labeled for 7 min at 26°C. Cells  were then chased at 26°C (A) or  shifted to 37°C (B) with prewarmed media and chased for  the indicated times. At each  time point, cells were separated  from the periplasmic fraction by  spheroplasting, lysates were prepared, and samples were immunoprecipitated with antiinvertase antibodies. Equal portions  of precipitate from each time  point were then separated on  8% SDS–polyacrylamide gels.  (C) Intracellular invertase from  the indicated time point was divided into three equal portions  and precipitated with antisera  against invertase, α1,6-mannose,  or α1,3-mannose residues as indicated. ER (core), Golgi (outerchain), and cytoplasmic (cyto.)  forms of invertase are indicated  in the left margin.
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Figure 4: ypt31-Δ/ypt32-A141D mutant cells exhibit a tight conditional secretory block of invertase transport downstream of the medial-Golgi. Wild-type (NSY349, left) and mutant (NSY348, right) cells were labeled for 7 min at 26°C. Cells were then chased at 26°C (A) or shifted to 37°C (B) with prewarmed media and chased for the indicated times. At each time point, cells were separated from the periplasmic fraction by spheroplasting, lysates were prepared, and samples were immunoprecipitated with antiinvertase antibodies. Equal portions of precipitate from each time point were then separated on 8% SDS–polyacrylamide gels. (C) Intracellular invertase from the indicated time point was divided into three equal portions and precipitated with antisera against invertase, α1,6-mannose, or α1,3-mannose residues as indicated. ER (core), Golgi (outerchain), and cytoplasmic (cyto.) forms of invertase are indicated in the left margin.

Mentions: Cells carrying the ypt32-A141D mutation and deleted for the YPT31 gene exhibit a tight temperature sensitivity for growth at 37°C (Fig. 3 B). We tested the effect of this mutation on the transport of two secreted proteins, invertase and α-factor, and a vacuolar protein, CPY. To determine the effect of the ypt31Δ/32-A141D (ypt31/32) mutation on protein transport at the permissive temperature, we compared the processing of invertase at 26°C in wildtype and mutant cells. Cells were labeled at 26°C for 7 min, and then chased for 10 or 30 min. Spheroplasts were separated from periplasmic contents, and invertase was immunoprecipitated from both fractions and analyzed by gel electrophoresis. It is possible to distinguish between invertase that resides in early secretory compartments (ER; core glycosylated and cis-Golgi; α-1,6-mannosylated), invertase in medial- or trans-Golgi (α-1,3-mannosylated), and secreted invertase (periplasmic). After 10 min of chase, wildtype cells secrete most (∼90%) of the invertase labeled during the pulse. ypt31/32 mutant cells also secrete most of the invertase (81%), which is fully glycosylated, but only after 30 min of chase (Fig. 4 A). Thus, the ypt31/32 mutant cells display a kinetic defect in secretion at the permissive temperature.


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 exhibit a tight conditional secretory block of invertase transport downstream of  the medial-Golgi. Wild-type  (NSY349, left) and mutant  (NSY348, right) cells were labeled for 7 min at 26°C. Cells  were then chased at 26°C (A) or  shifted to 37°C (B) with prewarmed media and chased for  the indicated times. At each  time point, cells were separated  from the periplasmic fraction by  spheroplasting, lysates were prepared, and samples were immunoprecipitated with antiinvertase antibodies. Equal portions  of precipitate from each time  point were then separated on  8% SDS–polyacrylamide gels.  (C) Intracellular invertase from  the indicated time point was divided into three equal portions  and precipitated with antisera  against invertase, α1,6-mannose,  or α1,3-mannose residues as indicated. ER (core), Golgi (outerchain), and cytoplasmic (cyto.)  forms of invertase are indicated  in the left margin.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: ypt31-Δ/ypt32-A141D mutant cells exhibit a tight conditional secretory block of invertase transport downstream of the medial-Golgi. Wild-type (NSY349, left) and mutant (NSY348, right) cells were labeled for 7 min at 26°C. Cells were then chased at 26°C (A) or shifted to 37°C (B) with prewarmed media and chased for the indicated times. At each time point, cells were separated from the periplasmic fraction by spheroplasting, lysates were prepared, and samples were immunoprecipitated with antiinvertase antibodies. Equal portions of precipitate from each time point were then separated on 8% SDS–polyacrylamide gels. (C) Intracellular invertase from the indicated time point was divided into three equal portions and precipitated with antisera against invertase, α1,6-mannose, or α1,3-mannose residues as indicated. ER (core), Golgi (outerchain), and cytoplasmic (cyto.) forms of invertase are indicated in the left margin.
Mentions: Cells carrying the ypt32-A141D mutation and deleted for the YPT31 gene exhibit a tight temperature sensitivity for growth at 37°C (Fig. 3 B). We tested the effect of this mutation on the transport of two secreted proteins, invertase and α-factor, and a vacuolar protein, CPY. To determine the effect of the ypt31Δ/32-A141D (ypt31/32) mutation on protein transport at the permissive temperature, we compared the processing of invertase at 26°C in wildtype and mutant cells. Cells were labeled at 26°C for 7 min, and then chased for 10 or 30 min. Spheroplasts were separated from periplasmic contents, and invertase was immunoprecipitated from both fractions and analyzed by gel electrophoresis. It is possible to distinguish between invertase that resides in early secretory compartments (ER; core glycosylated and cis-Golgi; α-1,6-mannosylated), invertase in medial- or trans-Golgi (α-1,3-mannosylated), and secreted invertase (periplasmic). After 10 min of chase, wildtype cells secrete most (∼90%) of the invertase labeled during the pulse. ypt31/32 mutant cells also secrete most of the invertase (81%), which is fully glycosylated, but only after 30 min of chase (Fig. 4 A). Thus, the ypt31/32 mutant cells display a kinetic defect in secretion at the permissive temperature.

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