Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers.
Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway.
Jones
S
S
Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637, USA.
Jedd
G
G
Kahn
R A
RA
Franzusoff
A
A
Bartolini
F
F
Segev
N
N
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000502:physiology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D005810:Multigene Family; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins
D005656:Fungal Proteins; C121632:GEA2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; C121631:SYT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C066755:Sec7 guanine nucleotide exchange factors; C087905:YPT31 protein, S cerevisiae; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C060755:YPT1 protein, S cerevisiae; C121633:YPT32 protein, S cerevisiae; D020727:ADP-Ribosylation Factors; D020691:rab GTP-Binding Proteins