Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.
In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley 94720, USA.
M J
MJ
Kuehn
R
R
Schekman
P O
PO
Ljungdahl
D018528:ATP-Binding Cassette Transporters; D026905:Amino Acid Transport Systems; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D001693:Biological Transport, Active; D022181:COP-Coated Vesicles; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D020690:GTPase-Activating Proteins; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D000072235:Mating Factor; D008565:Membrane Proteins / Q000494:pharmacology / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins; D028861:Nuclear Pore Complex Proteins; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013104:Spheroplasts / Q000378:metabolism; D033921:Vesicular Transport Proteins
D018528:ATP-Binding Cassette Transporters; D026905:Amino Acid Transport Systems; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D028861:Nuclear Pore Complex Proteins; C434364:PMA2 protein, S cerevisiae; D010455:Peptides; D011498:Protein Precursors; C079620:SEC13 protein, S cerevisiae; C079037:SEC23 protein, S cerevisiae; C077883:SHR3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D000072235:Mating Factor; C043828:histidine permease, Bacteria; D019204:GTP-Binding Proteins; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae