Palmitoylation of p59fyn is reversible and sufficient for plasma membrane association.
Members of the Src family of protein tyrosine kinases are localized to subspecialized regions of the plasma membrane. Herein we show that the N-terminal SH4 region of the Src family member p59fyn (Fyn) is both necessary and sufficient for targeting of Fyn and heterologous proteins to the plasma membrane and detergent-insoluble subdomains. Attachment of the first 16 amino acids of Fyn to a normally cytosolic protein, beta-galactosidase, resulted in distinct plasma membrane localization of the chimeric protein. Mutation of the palmitoylation site (cysteine-3) within Fyn16-beta-galactosidase or wild-type Fyn abrogated plasma membrane localization, resulting in redistribution of the mutant proteins into intracellular membranes. Substitution of the SH4 motif within Fyn with heterologous sequences from other palmitoylated proteins (G alpha o and GAP43) revealed that the presence of palmitate is sufficient to direct plasma membrane localization independent of surrounding amino acid sequences and myristate. Palmitoylated Fyn chimeras were also enriched in the Triton X-100-resistant matrix, whereas nonpalmitoylated forms of these proteins were detected in the detergent-soluble fraction. The palmitate moiety on Fyn exhibited a half-life of 1.5-2 h. In contrast, the half-life of the polypeptide backbone was 8 h, indicating that palmitoylation is a reversible modification. These studies establish that the palmitoylated SH4 sequence of Fyn can be used to specifically target proteins to the plasma membrane in a reversible manner.
Wolven
A
A
Program in Molecular Biology, Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.
Okamura
H
H
Rosenblatt
Y
Y
Resh
M D
MD
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D051379:Mice; D009226:Myristates / Q000378:metabolism; D017830:Octoxynol; D010168:Palmitates / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins
D009226:Myristates; D010168:Palmitates; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017830:Octoxynol; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn