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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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The YPT32-A141D  mutation is in a conserved  residue and confers a tight  conditional growth phenotype. (A) Comparison of the  amino acid sequence of the  α-helix 5 region in Ypt/rab  and ras proteins. Arrow indicates conserved residue, which  when mutated to Asp results  in temperature-sensitive function in Ypt1 (ypt1-A136D),  Ypt32 (ypt32-A141D), and  Sec4 (sec4-8, sec4-G147D).  (B) The ypt32-A141D mutation confers a temperaturesensitive growth phenotype  in cells deleted for the  YPT31 gene. Serial dilutions  of wild-type (NSY128), Δypt31  (NSY290), Δypt32 (NSY296),  and Δypt31,ypt32-A141D  (NSY313) cells were spotted  on YPD plates and incubated  at 26° or 37°C.
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Figure 3: The YPT32-A141D mutation is in a conserved residue and confers a tight conditional growth phenotype. (A) Comparison of the amino acid sequence of the α-helix 5 region in Ypt/rab and ras proteins. Arrow indicates conserved residue, which when mutated to Asp results in temperature-sensitive function in Ypt1 (ypt1-A136D), Ypt32 (ypt32-A141D), and Sec4 (sec4-8, sec4-G147D). (B) The ypt32-A141D mutation confers a temperaturesensitive growth phenotype in cells deleted for the YPT31 gene. Serial dilutions of wild-type (NSY128), Δypt31 (NSY290), Δypt32 (NSY296), and Δypt31,ypt32-A141D (NSY313) cells were spotted on YPD plates and incubated at 26° or 37°C.

Mentions: To determine whether the YPT31 and YPT32 genes are essential for viability, we studied the effect of their deletion on cell growth. YPT31 was deleted by precise replacement of its entire coding region with the HIS3 gene, and YPT32 by its precise replacement with either the kanr or the HIS3 gene. The replacement of both genes was confirmed by PCR analysis (not shown). Cells deleted for either YPT31 or YPT32 alone are viable (see Fig. 3 B). Thus, neither gene is essential for viability. The effect of deletion of YPT31 and/or YPT32 genes on cell growth was determined also by analyzing the ability of the cells to lose the YPT31 gene carried on a plasmid marked with URA3. Cells deleted for YPT31 and carrying the YPT31 gene on a plasmid can grow on 5-FOA plates, indicating that the cells can lose the plasmid (Fig. 1 C, top line). However, cells deleted for both YPT31 and YPT32 genes and carrying the YPT31 gene on a plasmid do not grow on 5-FOA plates, indicating that they cannot lose the YPT31 gene carried by the plasmid (Fig. 1 C, middle line). These cells can grow on 5-FOA plates if they contain in addition another plasmid (marked with LEU2) that carries the YPT31 gene (Fig. 1 C, bottom line). Together, these results show that while neither gene is required for cell growth, at least one of them is necessary for viability. These data and the high degree of homology between the two genes suggest that they are functional homologues or perform overlapping functions.


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

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

The YPT32-A141D  mutation is in a conserved  residue and confers a tight  conditional growth phenotype. (A) Comparison of the  amino acid sequence of the  α-helix 5 region in Ypt/rab  and ras proteins. Arrow indicates conserved residue, which  when mutated to Asp results  in temperature-sensitive function in Ypt1 (ypt1-A136D),  Ypt32 (ypt32-A141D), and  Sec4 (sec4-8, sec4-G147D).  (B) The ypt32-A141D mutation confers a temperaturesensitive growth phenotype  in cells deleted for the  YPT31 gene. Serial dilutions  of wild-type (NSY128), Δypt31  (NSY290), Δypt32 (NSY296),  and Δypt31,ypt32-A141D  (NSY313) cells were spotted  on YPD plates and incubated  at 26° or 37°C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139891&req=5

Figure 3: The YPT32-A141D mutation is in a conserved residue and confers a tight conditional growth phenotype. (A) Comparison of the amino acid sequence of the α-helix 5 region in Ypt/rab and ras proteins. Arrow indicates conserved residue, which when mutated to Asp results in temperature-sensitive function in Ypt1 (ypt1-A136D), Ypt32 (ypt32-A141D), and Sec4 (sec4-8, sec4-G147D). (B) The ypt32-A141D mutation confers a temperaturesensitive growth phenotype in cells deleted for the YPT31 gene. Serial dilutions of wild-type (NSY128), Δypt31 (NSY290), Δypt32 (NSY296), and Δypt31,ypt32-A141D (NSY313) cells were spotted on YPD plates and incubated at 26° or 37°C.
Mentions: To determine whether the YPT31 and YPT32 genes are essential for viability, we studied the effect of their deletion on cell growth. YPT31 was deleted by precise replacement of its entire coding region with the HIS3 gene, and YPT32 by its precise replacement with either the kanr or the HIS3 gene. The replacement of both genes was confirmed by PCR analysis (not shown). Cells deleted for either YPT31 or YPT32 alone are viable (see Fig. 3 B). Thus, neither gene is essential for viability. The effect of deletion of YPT31 and/or YPT32 genes on cell growth was determined also by analyzing the ability of the cells to lose the YPT31 gene carried on a plasmid marked with URA3. Cells deleted for YPT31 and carrying the YPT31 gene on a plasmid can grow on 5-FOA plates, indicating that the cells can lose the plasmid (Fig. 1 C, top line). However, cells deleted for both YPT31 and YPT32 genes and carrying the YPT31 gene on a plasmid do not grow on 5-FOA plates, indicating that they cannot lose the YPT31 gene carried by the plasmid (Fig. 1 C, middle line). These cells can grow on 5-FOA plates if they contain in addition another plasmid (marked with LEU2) that carries the YPT31 gene (Fig. 1 C, bottom line). Together, these results show that while neither gene is required for cell growth, at least one of them is necessary for viability. These data and the high degree of homology between the two genes suggest that they are functional homologues or perform overlapping functions.

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