A method for determining the in vivo topology of yeast polytopic membrane proteins demonstrates that Gap1p fully integrates into the membrane independently of Shr3p.
The general amino acid permease (Gap1p) of Saccharomyces cerevisiae is an integral membrane protein that contains 12 hydrophobic regions predicted to be membrane-spanning segments. A topological reporter construct, encoding an internal 53-amino acid peptide of invertase (Suc2p) containing three Asp-X-Ser/Thr glycosylation sites, was inserted in-frame into the hydrophilic NH(2)- and COOH-terminal domains and each of the 11 hydrophilic loops that separate the 12 hydrophobic segments of Gap1p. The resulting 13 gene sandwich fusion proteins were expressed in a gap1Delta null mutant strain; 9 of these retain amino acid transport activity and are folded and correctly targeted to the plasma membrane. The glycosylation state of each of the fusion proteins was monitored; the results indicate that all 12 hydrophobic segments of Gap1p span the membrane, and the NH(2) and COOH termini are cytoplasmically oriented. These results were independently tested by isolating sealed right-side-out microsomes from sec12-1 strains expressing six different Gap1p constructs containing functional factor Xa protease cleavage sites. The pattern of factor Xa protease cleavage was found to be consistent with the presence of 12 membrane-spanning domains. Gap1p exhibited the same membrane topology in strains lacking Shr3p; therefore, Gap1p fully integrates into the ER membrane independently of this permease-specific packaging chaperone.
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Ludwig Institute for Cancer Research, Box 240, S-171 77 Stockholm, Sweden.
Ljungdahl
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D026905:Amino Acid Transport Systems; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D019286:Endopeptidase K / Q000494:pharmacology; D015951:Factor Xa / Q000494:pharmacology; D017930:Genes, Reporter; D006031:Glycosylation; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
D026905:Amino Acid Transport Systems; D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C077883:SHR3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; C078729:sucrose transport protein, plant; D015951:Factor Xa; D019286:Endopeptidase K