Reconstitution of retrograde transport from the Golgi to the ER in vitro.
Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone alpha-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369-377). Retrieval depends on the HDEL sequence; the alpha-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.
Spang
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Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
Schekman
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D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000251:Adenosine Triphosphatases; D001693:Biological Transport, Active; D018699:Coated Vesicles; D020756:Coatomer Protein; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D020756:Coatomer Protein; D005656:Fungal Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C077480:histidyl-aspartyl-glutamyl-leucine; D000251:Adenosine Triphosphatases; D019204:GTP-Binding Proteins; C064404:SEC18 protein, S cerevisiae; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors