Membrane association of the myristoylated alanine-rich C kinase substrate (MARCKS) protein appears to involve myristate-dependent binding in the absence of a myristoyl protein receptor.

The myristoylated alanine-rich C kinase substrate, or MARCKS protein, has been implicated in several cellular processes, yet its physiological function remains unknown. We have studied the molecular basis of its membrane association in a cell-free system in order to help elucidate its regulation and function. First, we showed that the MARCKS protein incorporated [3H]myristate when its mRNA was translated in vitro in reticulocyte lysates. The myristoylated protein bound rapidly to freshly fractionated cell membranes, while a nonmyristoylated mutant associated to a much lesser extent (< 15% of wild type). To determine whether this binding was due to a specific cytoplasmic-face protein "receptor," as is seen with pp60v-src, we pretreated the membranes in several ways. Prior treatment of membranes with heat (100 degrees C for 3 min) or trypsin did not affect subsequent MARCKS binding. Binding was markedly decreased in 50 mM EDTA, 0.5 M NaCl, or 1.0% Triton X-100; it was restored to normal after removal of the NaCl and EDTA but was still decreased after removal of the Triton X-100. These findings argued against the existence of a protein receptor for the MARCKS protein on cellular membranes. Finally, MARCKS protein phosphorylated in vitro with protein kinase C bound to the cell membranes to the same extent as the nonphosphorylated protein; this binding was also unaffected by an excess of a synthetic peptide corresponding to the phosphorylation site domain of the protein. We conclude that, at least in this in vitro system, the membrane association of the MARCKS protein is primarily dependent on the amino-terminal myristate moiety and does not appear to involve a specific cytoplasmic-face protein receptor.