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Cell Biology and Genetics Program, Sloan-Kettering Institute for Cancer Research, New York, 10021
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
o-, Gs-, 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.
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