Analysis of the site occupancy constraints of primary amino acid sequencesin the motif directing palmitylation of the vaccinia virus 37-kDa envelopeprotein

Vaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmity lated on cysteine residues 185 and 186 of the 372-amino acid protein. We ha ve previously reported on a loosely conserved consensus motif. Further anal ysis has identified a conserved consensus sequence, Hydro*AAC(C)A (Hydro* r epresents a hydrophobic portion of a protein determined by any one of the f ollowing: a hydrophobic sequence, a transmembrane domain 1-12 amino acids a way from the modification site, or the prior addition of a hydrophobic mole cule; C, palmitate acceptor cysteines; A aliphatic residue) that is respons ible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstream and downstream of the palmitate acceptor residues in p37 by site-directed mutagenesis and transi ent expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183 and 184 of the wild-type p37 allo w for efficient palmitylation. In contrast, the replacement of leucine at p osition 187 with glycine increases palmitylation efficiency. The 10 amino a cids immediately upstream of the palmitate acceptor site are absolutely nec essary while the downstream 10 aminoacids are dispensable. These results to gether with previous data suggests that the Hydro*AAC(C)A motif is required for efficient palmitylation of p37. (C) 1999 Academic Press.