The immunodetected yeast purine-cytosine permease is not N-linked glycosylated, nor are glycosylation sequences required to have a functional permease.
The purine-cytosine permease (PCP) of the yeast Saccharomyces cerevisiae was detected by immunological methods. Using antibodies directed against synthetic peptides, whose sequences were derived from the primary structure of the PCP, immunoprecipitation of [35S]methionine-labelled PCP was achieved either from cellular extracts or from in vitro translation mixtures. Non-labelled PCP was also detected on Western blots of membrane proteins. Similar migration rates were observed for PCP originating both from immunoprecipitated cellular extracts and from in vitro translation mixtures. Hence, post-translational processing, if any, only slightly affects the size of the protein. Also no evidence was found for N-linked core-glycosylation: identical migration rates were observed when immunoprecipitated PCP molecules were extracted from cells labelled for 10 min with [35S]methionine, pretreated or not with tunicamycin. On the other hand, the suppression of the two potential N-linked glycosylation sequences in the DNA did not lead to inactivation of the transport activity, confirming that N-linked glycosylation is not required for the permease activity.
Rodriguez
C
C
URA-GEM (D1481), Institut de Botanique, Strasbourg, France.
Bloch
J C
JC
Chevallier
M R
MR
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003596:Cytosine / Q000378:metabolism; D006031:Glycosylation; D015151:Immunoblotting; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033702:Nucleobase Transport Proteins; D017354:Point Mutation; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; C043181:FCY2 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D003596:Cytosine