Rapid plasma membrane anchoring of newly synthesized p59fyn: selective requirement for NH2-terminal myristoylation and palmitoylation at cysteine-3.
The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59fyn. Metabolic labeling of transfected COS or NIH 3T3 cells with [35S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20-60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1-2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH2-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of G alpha(o)-, G alpha(s)-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10-20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.
van't Hof
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Cell Biology and Genetics Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10021, USA.
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D016475:3T3 Cells; D000215:Acylation; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D003902:Detergents; D019922:GAP-43 Protein; D019204:GTP-Binding Proteins / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D009154:Mutation; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics; D017830:Octoxynol; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D011132:Polyribosomes / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D003902:Detergents; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009227:Myristic Acids; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D019308:Palmitic Acid; D017830:Octoxynol; C494957:FYN protein, human; C494960:Fyn protein, mouse; D015688:Oncogene Protein pp60(v-src); D051076:Proto-Oncogene Proteins c-fyn; D016392:Proto-Oncogene Proteins pp60(c-src); D019204:GTP-Binding Proteins; D003545:Cysteine