A C-terminal sequence in the guanine nucleotide exchange factor Sec7 mediates Golgi association and interaction with the Rsp5 ubiquitin ligase.
Arf GTPases control vesicle formation from different intracellular membranes and are regulated by Arf guanine nucleotide exchange factors (GEFs). Outside of their conserved catalytic domains, known as Sec7 domains, little is known about Arf GEFs. Rsp5 is a yeast ubiquitin ligase that regulates numerous membrane trafficking events and carries a C2 domain that is specifically required for trans-Golgi network to vacuole transport. In a screen for proteins that interact with the Rsp5 C2 domain we identified Sec7, the GEF that acts on Golgi-associated Arfs. The Rsp5-Sec7 interaction is direct, occurs in vivo, and is conserved among mammalian Rsp5 and Sec7 homologues. A 50-amino acid region near the Sec7 C terminus is required for Rsp5 binding and for normal Sec7 localization. Binding of Sec7 to Rsp5 is dependent on the presence of the phosphoinositide 3-kinase Vps34, suggesting that phosphatidylinositol 3-phosphate (PI(3)P) plays a role in regulating this interaction. Overexpression of Sec7 significantly suppresses the growth and sorting defects of an rsp5 C2 domain point mutant. These observations identify a new functional region within the Sec7/BIG family of Arf GEFs that is required for trans-Golgi network localization.
Dehring
Deborah A Klos
DA
Department of Biochemistry, Molecular Biology & Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Adler
Adam S
AS
Hosseini
Ava
A
Hicke
Linda
L
D002384:Catalysis; D056827:Endosomal Sorting Complexes Required for Transport; D005838:Genotype; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000502:physiology*; D008856:Microscopy, Fluorescence; D010641:Phenotype; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D020798:Two-Hybrid System Techniques; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry*
D056827:Endosomal Sorting Complexes Required for Transport; D020662:Guanine Nucleotide Exchange Factors; D018129:Phosphatidylinositol Phosphates; D029701:Saccharomyces cerevisiae Proteins; C066755:Sec7 guanine nucleotide exchange factors; C055525:phosphatidylinositol 3-phosphate; D049452:Green Fluorescent Proteins; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae