A carboxyl-terminal cysteine residue is required for palmitic acid binding and biological activity of the ras-related yeast YPT1 protein.

Abstract

The Saccharomyces cerevisiae YPT1 gene codes for a ras‐like, guanine nucleotide‐binding protein which is essential for cell viability. The functional significance of two consecutive cysteines at the very carboxyl‐terminal end of this protein and in ypt homologues of other eukaryotic species was examined. YPT1 gene mutations were generated that either led to substitutions by serine or the deletion of one or both C‐terminal cysteines. The consequences of the mutations were checked in cells after replacing the wild type with the mutant genes. It was found that as long as one of the cysteines was retained, the protein was fully functional. The YPT1 protein could be labelled with [3H]palmitic acid that appeared to be bound in an ester linkage. The wild‐type protein was evenly distributed between soluble and membrane‐associated proteins, the palmitoylated form was predominantly in the crude membrane fraction. The mutant protein lacking the C‐terminal cysteines was not palmitoylated and was exclusively found in the soluble fraction. The extension by three residues, ‐Val‐Leu‐Ser, generating a ras‐typical C‐terminal end, did not interfere with the mutant YPT1 protein's function although it resulted in a reduced labelling with palmitic acid.

The ras-like protein p25rab3A is partially cytosolic and is expressed only in neural tissue.

“…The correct C-terminal sequence is essential for processing and membrane insertion of p2lras, which is cleaved, carboxymethylated, isoprenylated, and palmitoylated (9). The two consecutive Cys residues of rabi (YPT1) are also essential for acylation and membrane association (14). The observation that p25rab3A is partially cytosolic, like YPT1 (14), therefore supports the idea that the different c-terminal consensus sequences comprise signals for different types of processing and localization.…”

“…These levels of contamination cannot account for the levels of p25rab3A detected in the membrane fractions (116% of cytosolic p25rab3A in the detergent-solubilized particulate fraction; 30% in the purified membranes). The rab3A gene product, like YPT1 (rabl) (14), therefore appears to be partially membrane bound and partially cytosolic. DISCUSSION We have prepared an antipeptide antiserum that specifically detects the GTP-binding protein p25rab3A and have shown that this protein is expressed exclusively in brain tissue of the tissues tested.…”

The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.

“…Ras-related proteins are involved in regulating a wide range of cellular processes, including cell proliferation, differentiation, intracellular trafficking (7, 24), membrane ruffling, and assembly of actin stress fibers (43,44). Although these proteins serve very diverse functions, their ability to associate with cell membranes is a fundamental requirement for biological activity (review in reference 20) such that mutations in Ras, Rho, and Rab proteins which block membrane localization also render these proteins biologically inactive (2,21,40,49,51).The mechanism of attachment of Ras proteins to the inner surface of the plasma membrane provides a model for the membrane targeting of other Ras-related proteins. The C terminus of Ras terminates in a CAAX motif (C = cysteine, A = aliphatic amino acid, X = any amino acid) which undergoes farnesylation of the cysteine residue (12, 28), proteolysis by an endopeptidase to remove the AAX amino acids (19, 23), and carboxyl-methyl esterification of the cysteine (16,23 (510) 222 9758. signal is a polylysine domain in K-Ras and cysteine palmitoylation sites in the H-and N-Ras proteins.…”

“…Ras-related proteins are involved in regulating a wide range of cellular processes, including cell proliferation, differentiation, intracellular trafficking (7, 24), membrane ruffling, and assembly of actin stress fibers (43,44). Although these proteins serve very diverse functions, their ability to associate with cell membranes is a fundamental requirement for biological activity (review in reference 20) such that mutations in Ras, Rho, and Rab proteins which block membrane localization also render these proteins biologically inactive (2,21,40,49,51).…”

C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

“…Less is known about the posttranslational processing of proteins in the second and third groups. A member of the second group of proteins, the yeast YPT1 protein, has been reported to be palmitoylated on one or both of the cysteine residues at the C-terminal region, but this result has not been substantiated (11). In the case of the small G proteins in the third group, no information concerning their posttranslational modification is available.…”