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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 YPT31 and YPT32 genes encode two functionally homologous exocytic GTPases. (A) Comparison of the amino acid sequences of Ypt31 and Ypt32 proteins. The predicted protein sequence of Ypt32 was compared to Ypt31 using the MegAlign program  (DNAStar Inc., Madison, WI, Clustal method with the PAM250 residue weight table). Identities are shaded with solid black, and residues conserved to within two distance units are shaded. Overall, the two proteins are 81.1% identical and 89.6% similar when compared  using the bestfit program (Genetics Computer Group, Madison, WI). (B) Ypt31 and Ypt32 proteins belong to a subfamily of exocytic Ypt  GTPases by phylogenetic analysis of the Ypt/rab family of small GTPases. The predicted amino acid sequence of Ypt31 and Ypt32 were  compared to all other Ypt proteins, using the completed S. cerevisiae genome sequence and their human homologues. The analysis  shows that Ypt/rab proteins fall into two functional subfamilies: those involved in endocytosis and vacuolar protein sorting (Endocytosis-Vac.) and those involved in exocytosis. Sequences were aligned as above. The scale at the bottom indicates the number of substitutions  between sequences. hum., Homo sapiens; H-ras, Harvey murine sarcoma virus ras protein; all other sequences, Saccharomyces cerevisiae.  These sequence data are available from GenBank/EMBL/DDBJ under accession numbers: Ypt31, U18778; Ypt32, X72834; rab11, X56740;  Ypt7, X68144; rab7, U44104; Ypt1, X00209; rab1a, M28209; Sec4, M16507; Ypt6, U17244; rab6a, M28212; Ypt51, X76173; Ypt52,  X76174; Ypt53, X76175; rab5b, X54871; and H-ras, X00740. (C) Requirement of YPT31 or YPT32 genes for cell viability. The YPT31  gene was precisely deleted using the HIS3 gene, and this strain was transformed with a URA3-marked CEN vector containing the  YPT31 gene under control of its own promoter (NSY301, first row). This strain (NSY301) was subsequently deleted for the YPT32 gene  using the KANr gene as a dominant delectable marker (NSY302, middle row). Finally, this strain (NSY302) was transformed with a second plasmid marked with LEU2 and carrying the YPT31 gene (NSY306, bottom row). The three strains were grown in synthetic media  maintaining selection for plasmids. Serial dilutions of cells were then spotted onto either SD or SD-FOA and grown at 26°C. Cells deleted for both genes do not grow on SD-FOA plates, indicating that they cannot lose the URA-marked YPT31 plasmid unless they carry  also the LEU-marked YPT31 plasmid.
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Figure 1: The YPT31 and YPT32 genes encode two functionally homologous exocytic GTPases. (A) Comparison of the amino acid sequences of Ypt31 and Ypt32 proteins. The predicted protein sequence of Ypt32 was compared to Ypt31 using the MegAlign program (DNAStar Inc., Madison, WI, Clustal method with the PAM250 residue weight table). Identities are shaded with solid black, and residues conserved to within two distance units are shaded. Overall, the two proteins are 81.1% identical and 89.6% similar when compared using the bestfit program (Genetics Computer Group, Madison, WI). (B) Ypt31 and Ypt32 proteins belong to a subfamily of exocytic Ypt GTPases by phylogenetic analysis of the Ypt/rab family of small GTPases. The predicted amino acid sequence of Ypt31 and Ypt32 were compared to all other Ypt proteins, using the completed S. cerevisiae genome sequence and their human homologues. The analysis shows that Ypt/rab proteins fall into two functional subfamilies: those involved in endocytosis and vacuolar protein sorting (Endocytosis-Vac.) and those involved in exocytosis. Sequences were aligned as above. The scale at the bottom indicates the number of substitutions between sequences. hum., Homo sapiens; H-ras, Harvey murine sarcoma virus ras protein; all other sequences, Saccharomyces cerevisiae. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers: Ypt31, U18778; Ypt32, X72834; rab11, X56740; Ypt7, X68144; rab7, U44104; Ypt1, X00209; rab1a, M28209; Sec4, M16507; Ypt6, U17244; rab6a, M28212; Ypt51, X76173; Ypt52, X76174; Ypt53, X76175; rab5b, X54871; and H-ras, X00740. (C) Requirement of YPT31 or YPT32 genes for cell viability. The YPT31 gene was precisely deleted using the HIS3 gene, and this strain was transformed with a URA3-marked CEN vector containing the YPT31 gene under control of its own promoter (NSY301, first row). This strain (NSY301) was subsequently deleted for the YPT32 gene using the KANr gene as a dominant delectable marker (NSY302, middle row). Finally, this strain (NSY302) was transformed with a second plasmid marked with LEU2 and carrying the YPT31 gene (NSY306, bottom row). The three strains were grown in synthetic media maintaining selection for plasmids. Serial dilutions of cells were then spotted onto either SD or SD-FOA and grown at 26°C. Cells deleted for both genes do not grow on SD-FOA plates, indicating that they cannot lose the URA-marked YPT31 plasmid unless they carry also the LEU-marked YPT31 plasmid.

Mentions: We have found two new YPT genes as high-copy suppressors of the dominant YPT1D124N mutation (Jones et al., 1995; Jones, S., and N. Segev, manuscript in preparation). The sequences of the two genes revealed that they belong to the Ypt/rab family of GTPases and that their protein products share 81% identity and 90% similarity (Fig. 1 A). These genes were submitted to the yeast database by other researchers as YPT31 or YPT8 (Lai et al., 1994) and YPT32. (These sequence data are available from GenBank/EMBL/ DDBJ under accession numbers U18778 and X72834, respectively.) The high degree of sequence similarity between the two genes suggested that they might be functionally homologous. The protein products are most similar to the mammalian rab11 GTPase and to Schizosaccharomyces pombe Ypt3 (62 and 63% identity, respectively), and their closest Saccharomyces cerevisiae homologues are Ypt1 and Sec4 (42–46% identity; Ypt1 and Sec4 share 48% identity). Phylogenetic analysis of all Ypt proteins present in S. cerevisiae, using the completed genome sequence available for this organism, and of their human homologues reveals that Ypt/rab GTPases fall into two subfamilies: exocytic and endocytic/vacuolar. The two new Ypt proteins, Ypt31 and Ypt32, seem to belong to the subfamily of exocytic Ypt/rab GTPases (Fig. 1 B). We wished to study the possible role of Ypt31 and Ypt32 proteins in the yeast exocytic pathway since very little is known about components that regulate the secretory steps between those regulated by the Ypt1 and Sec4 GTPases.


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 YPT31 and YPT32 genes encode two functionally homologous exocytic GTPases. (A) Comparison of the amino acid sequences of Ypt31 and Ypt32 proteins. The predicted protein sequence of Ypt32 was compared to Ypt31 using the MegAlign program  (DNAStar Inc., Madison, WI, Clustal method with the PAM250 residue weight table). Identities are shaded with solid black, and residues conserved to within two distance units are shaded. Overall, the two proteins are 81.1% identical and 89.6% similar when compared  using the bestfit program (Genetics Computer Group, Madison, WI). (B) Ypt31 and Ypt32 proteins belong to a subfamily of exocytic Ypt  GTPases by phylogenetic analysis of the Ypt/rab family of small GTPases. The predicted amino acid sequence of Ypt31 and Ypt32 were  compared to all other Ypt proteins, using the completed S. cerevisiae genome sequence and their human homologues. The analysis  shows that Ypt/rab proteins fall into two functional subfamilies: those involved in endocytosis and vacuolar protein sorting (Endocytosis-Vac.) and those involved in exocytosis. Sequences were aligned as above. The scale at the bottom indicates the number of substitutions  between sequences. hum., Homo sapiens; H-ras, Harvey murine sarcoma virus ras protein; all other sequences, Saccharomyces cerevisiae.  These sequence data are available from GenBank/EMBL/DDBJ under accession numbers: Ypt31, U18778; Ypt32, X72834; rab11, X56740;  Ypt7, X68144; rab7, U44104; Ypt1, X00209; rab1a, M28209; Sec4, M16507; Ypt6, U17244; rab6a, M28212; Ypt51, X76173; Ypt52,  X76174; Ypt53, X76175; rab5b, X54871; and H-ras, X00740. (C) Requirement of YPT31 or YPT32 genes for cell viability. The YPT31  gene was precisely deleted using the HIS3 gene, and this strain was transformed with a URA3-marked CEN vector containing the  YPT31 gene under control of its own promoter (NSY301, first row). This strain (NSY301) was subsequently deleted for the YPT32 gene  using the KANr gene as a dominant delectable marker (NSY302, middle row). Finally, this strain (NSY302) was transformed with a second plasmid marked with LEU2 and carrying the YPT31 gene (NSY306, bottom row). The three strains were grown in synthetic media  maintaining selection for plasmids. Serial dilutions of cells were then spotted onto either SD or SD-FOA and grown at 26°C. Cells deleted for both genes do not grow on SD-FOA plates, indicating that they cannot lose the URA-marked YPT31 plasmid unless they carry  also the LEU-marked YPT31 plasmid.
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Related In: Results  -  Collection

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

Figure 1: The YPT31 and YPT32 genes encode two functionally homologous exocytic GTPases. (A) Comparison of the amino acid sequences of Ypt31 and Ypt32 proteins. The predicted protein sequence of Ypt32 was compared to Ypt31 using the MegAlign program (DNAStar Inc., Madison, WI, Clustal method with the PAM250 residue weight table). Identities are shaded with solid black, and residues conserved to within two distance units are shaded. Overall, the two proteins are 81.1% identical and 89.6% similar when compared using the bestfit program (Genetics Computer Group, Madison, WI). (B) Ypt31 and Ypt32 proteins belong to a subfamily of exocytic Ypt GTPases by phylogenetic analysis of the Ypt/rab family of small GTPases. The predicted amino acid sequence of Ypt31 and Ypt32 were compared to all other Ypt proteins, using the completed S. cerevisiae genome sequence and their human homologues. The analysis shows that Ypt/rab proteins fall into two functional subfamilies: those involved in endocytosis and vacuolar protein sorting (Endocytosis-Vac.) and those involved in exocytosis. Sequences were aligned as above. The scale at the bottom indicates the number of substitutions between sequences. hum., Homo sapiens; H-ras, Harvey murine sarcoma virus ras protein; all other sequences, Saccharomyces cerevisiae. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers: Ypt31, U18778; Ypt32, X72834; rab11, X56740; Ypt7, X68144; rab7, U44104; Ypt1, X00209; rab1a, M28209; Sec4, M16507; Ypt6, U17244; rab6a, M28212; Ypt51, X76173; Ypt52, X76174; Ypt53, X76175; rab5b, X54871; and H-ras, X00740. (C) Requirement of YPT31 or YPT32 genes for cell viability. The YPT31 gene was precisely deleted using the HIS3 gene, and this strain was transformed with a URA3-marked CEN vector containing the YPT31 gene under control of its own promoter (NSY301, first row). This strain (NSY301) was subsequently deleted for the YPT32 gene using the KANr gene as a dominant delectable marker (NSY302, middle row). Finally, this strain (NSY302) was transformed with a second plasmid marked with LEU2 and carrying the YPT31 gene (NSY306, bottom row). The three strains were grown in synthetic media maintaining selection for plasmids. Serial dilutions of cells were then spotted onto either SD or SD-FOA and grown at 26°C. Cells deleted for both genes do not grow on SD-FOA plates, indicating that they cannot lose the URA-marked YPT31 plasmid unless they carry also the LEU-marked YPT31 plasmid.
Mentions: We have found two new YPT genes as high-copy suppressors of the dominant YPT1D124N mutation (Jones et al., 1995; Jones, S., and N. Segev, manuscript in preparation). The sequences of the two genes revealed that they belong to the Ypt/rab family of GTPases and that their protein products share 81% identity and 90% similarity (Fig. 1 A). These genes were submitted to the yeast database by other researchers as YPT31 or YPT8 (Lai et al., 1994) and YPT32. (These sequence data are available from GenBank/EMBL/ DDBJ under accession numbers U18778 and X72834, respectively.) The high degree of sequence similarity between the two genes suggested that they might be functionally homologous. The protein products are most similar to the mammalian rab11 GTPase and to Schizosaccharomyces pombe Ypt3 (62 and 63% identity, respectively), and their closest Saccharomyces cerevisiae homologues are Ypt1 and Sec4 (42–46% identity; Ypt1 and Sec4 share 48% identity). Phylogenetic analysis of all Ypt proteins present in S. cerevisiae, using the completed genome sequence available for this organism, and of their human homologues reveals that Ypt/rab GTPases fall into two subfamilies: exocytic and endocytic/vacuolar. The two new Ypt proteins, Ypt31 and Ypt32, seem to belong to the subfamily of exocytic Ypt/rab GTPases (Fig. 1 B). We wished to study the possible role of Ypt31 and Ypt32 proteins in the yeast exocytic pathway since very little is known about components that regulate the secretory steps between those regulated by the Ypt1 and Sec4 GTPases.

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