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The covalent attachment of fatty acids - commonly palmitic acid - in a thioester-type linkage is a widespread modification of viral and cellular polypeptides. With integral membrane proteins the hydrocarbon chain is usually bound to cysteine residues located close to the boundary between the transmembrane region and the cytoplasmic tail. Inspection of the amino acids in the vicinity of the acylated cysteine residues reveals no obvious ‘consensus-signal’ for palmitoylation even if introduction of cysteine residues into the potential acylation region of a normally non-acylated glycoprotein is not sufficient for attachment of fatty acids. This implies that acylated membrane proteins contain complex conformational signals for palmitoylation that are mainly located within the cytoplasmic half of the transmembrane domain, but also involve the C-tail region. Using a series of new chimeric mutant proteins derived from acylated proteins as Influenza virus haemagglutinin as well as CD4 and CD8 receptor protein and from the non-acylated Sendai virus fusion protein we wanted to demonstrate the influence of transmembrane and cytoplasmic region for palmitoylation as well as the particular contribution of glycine residues within the TM-domain.
Sequence alignment of some palmitoylated proteins shows frequent occurrence of glycine in the transmembrane region close to the cytoplasmic tail. Hydrophobic glycine residues on the hydrophilic face of the TMD-helix may support palmitoylation of membrane proteins. While Sendai virus fusion protein is not palmitoylated even after introducing of a cysteine residue into the potential acylation region, substitution of the transmembrane domain or both transmembrane and cytoplasmic region for the corresponding regions of Influenza virus haemagglutinin or CD4- or CD8 receptor protein is sufficient for palmitoylation. Replacement of cytoplasma-near glycine and phenylalanine residues in the transmembrane region leads to a lower acylation rate of the chimerae, while distant glycine residues have no significant influence on palmitoylation rate. Our data suggest that in the absence of any consensus sequence there are still molecular features present in the palmitoylated area in viral and cellular transmembrane proteins which determine attachment of fatty acids. Possibly, these need to be recognized by a putative palmitoyltransferase (PAT). The protein segments located in the cytoplasmic regions of the transmembrane domain are of particular significance. Hydrophobic phenylalanine and especially glycine appear to be important for palmitoylation due to influence on the α-helix in the membrane.