Mutations in a highly conserved region of the Arf1p activator GEA2 block anterograde Golgi transport but not COPI recruitment to membranes.
We have identified an important functional region of the yeast Arf1 activator Gea2p upstream of the catalytic Sec7 domain and characterized a set of temperature-sensitive (ts) mutants with amino acid substitutions in this region. These gea2-ts mutants block or slow transport of proteins traversing the secretory pathway at exit from the endoplasmic reticulum (ER) and the early Golgi, and accumulate both ER and early Golgi membranes. No defects in two types of retrograde trafficking/sorting assays were observed. We find that a substantial amount of COPI is associated with Golgi membranes in the gea2-ts mutants, even after prolonged incubation at the nonpermissive temperature. COPI in these mutants is released from Golgi membranes by brefeldin A, a drug that binds directly to Gea2p and blocks Arf1 activation. Our results demonstrate that COPI function in sorting of at least three retrograde cargo proteins within the Golgi is not perturbed in these mutants, but that forward transport is severely inhibited. Hence this region of Gea2p upstream of the Sec7 domain plays a role in anterograde transport that is independent of its role in recruiting COPI for retrograde transport, at least of a subset of Golgi-ER cargo.
Park
Sei-Kyoung
SK
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Hartnell
Lisa M
LM
Jackson
Catherine L
CL
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000595:Amino Acid Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D020755:Coat Protein Complex I / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006056:Golgi Apparatus / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D020755:Coat Protein Complex I; C121632:GEA2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D029701:Saccharomyces cerevisiae Proteins; D020823:ADP-Ribosylation Factor 1