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Biochemistry

Title:		Partial constitutive activation of pheromone responses by a palmitoylation-site mutant of a G protein alpha subunit in yeast
Author(s):		Song J; Dohlman HG;
Address:		"Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA"
Journal Title:		Biochemistry
Year:		1996
Volume:		35
Issue:		47
Page Number:		14806 - 14817
DOI:		10.1021/bi961846b
ISSN/ISBN:		0006-2960 (Print) 0006-2960 (Linking)
Abstract:		"G protein alpha subunits are often myristoylated and/or palmitoylated near their amino terminus. The G protein alpha subunit in the yeast Saccharomyces cerevisiae (GPA1 gene product, Gpa1p) is known to be myristoylated, and this modification is essential for G protein activity in vivo. Here we examined whether Gpa1p is palmitoylated and determined the functional consequences of this modification. [3H]-Palmitic acid was incorporated into Gpa1p in cells expressing myc-tagged Gpa1p or Gpa1p-Gst. The label was released upon hydroxylamine treatment. Substitution of the conserved Cys 3 for Ser blocked incorporation of the label (Gpa1pC3S). Palmitoylation was also blocked by a mutation that prevents myristoylation (Gly2Ala), whereas the palmitoylation-site mutation had no effect on myristoylation of Gpa1p. Gpa1pC3S complemented the gpa1 delta mutation in vivo and formed a complex with G beta gamma that was able to undergo nucleotide exchange in vitro. However, basal and pheromone-induced FUSl-lacZ transcription were 2-5-fold higher in the C3S mutant. Pheromone-induced growth arrest was also enhanced by the mutation, but recovery from arrest was not affected. Like wild-type Gpa1p, the C3S mutant was predominantly membrane-associated. Upon Triton X-114 partitioning or high pH treatment, no difference in the membrane-binding properties of the wild-type Gpa1p and the C3S mutant was detected. By sucrose density gradient centrifugation of membranes, however, most of the mutant protein was mislocalized to a non-plasma membrane compartment, whereas G beta gamma localization was unaltered. Taken together, our data suggest that Gpa1p is palmitoylated via a thioester bond at Cys 3 and that palmitoylation plays a role in modulating Gpa1p signaling and membrane localization"
Keywords:		"Cell Membrane/metabolism Cysteine/metabolism Esters/metabolism Fungal Proteins/genetics/metabolism *GTP-Binding Protein alpha Subunits GTP-Binding Protein alpha Subunits, Gq-G11 GTP-Binding Proteins/genetics/*metabolism Guanosine Diphosphate/metabolism *H;"
Notes:		"MedlineSong, J Dohlman, H G eng Research Support, Non-U.S. Gov't 1996/11/26 Biochemistry. 1996 Nov 26; 35(47):14806-17. doi: 10.1021/bi961846b"