The GTPase ARF1p controls the sequence-specific vacuolar sorting route to the lytic vacuole.
We have studied the transport of soluble cargo molecules by inhibiting specific transport steps to and from the Golgi apparatus. Inhibition of export from the Golgi via coexpression of a dominant-negative GTP-restricted ARF1 mutant (Q71L) inhibits the secretion of alpha-amylase and simultaneously induces the secretion of the vacuolar protein phytepsin to the culture medium. By contrast, specific inhibition of endoplasmic reticulum export via overexpression of Sec12p or coexpression of a GTP-restricted form of Sar1p inhibits the anterograde transport of either cargo molecule in a similar manner. Increased secretion of the vacuolar protein was not observed after incubation with the drug brefeldin A or after coexpression of the GDP-restricted mutant of ARF1 (T31N). Therefore, the differential effect of inducing the secretion of one cargo molecule while inhibiting the secretion of another is dependent on the GTP hydrolysis by ARF1p and is not caused by a general inhibition of Golgi-derived COPI vesicle traffic. Moreover, we demonstrate that GTP-restricted ARF1-stimulated secretion is observed only for cargo molecules that are expected to be sorted in a BP80-dependent manner, exhibiting sequence-specific, context-independent, vacuolar sorting signals. Induced secretion of proteins carrying C-terminal vacuolar sorting signals was not observed. This finding suggests that ARF1p influences the BP80-mediated transport route to the vacuole in addition to transport steps of the default secretory pathway to the cell surface.
Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, United Kingdom.
Peter
P
Pimpl
Sally L
SL
Hanton
J Philip
JP
Taylor
Luis L
LL
Pinto-daSilva
Jürgen
J
Denecke
D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D022181:COP-Coated Vesicles / Q000187:drug effects / Q000378:metabolism; D020755:Coat Protein Complex I / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D021381:Protein Transport / Q000187:drug effects; D013379:Substrate Specificity; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D020755:Coat Protein Complex I; D020126:Brefeldin A; D020558:GTP Phosphohydrolases; D020823:ADP-Ribosylation Factor 1; D000077191:Wortmannin