Abstract
Triton X-114 phase partitioning, a procedure used for purifying integral membrane proteins, was used to study protein components of the mammalian visual transduction cascade. An integral membrane protein, rhodopsin, and two isoprenylated protein complexes, cyclic GMP phosphodiesterase and Gt beta gamma, partitioned into the detergent-rich phase. Arrestin, a soluble protein, accumulated in the aqueous phase. Gt alpha distributed about equally between phases whether GDP (Gt alpha.GDP) or GTP (Gt alpha.GTP) was bound. Gt beta gamma increased recovery of Gt alpha.GDP but not Gt alpha.GTP in the detergent phase. Trypsin-treated Gt alpha, which lacks the fatty acylated amino-terminal 2-kDa region, accumulated to a greater extent in the aqueous phase than did intact Gt alpha. Trypsinized cGMP phosphodiesterase, which lacks the isoprenyl group, partitioned into the aqueous phase. A carboxyl-terminal truncated mutant (Val-331 stop) of Gt alpha accumulated more in the aqueous phase then did recombinant full-length Gt alpha, supporting the role of the carboxyl terminus in increasing its hydrophobicity. N-Myristoylated recombinant Go alpha was more hydrophobic than recombinant Go alpha without myristate. ADP-ribosylation of Gt alpha catalyzed by NAD:arginine ADP-ribosyltransferase, but not by pertussis toxin, increased hydrophilicity. Triton X-114 phase partitioning can thus semiquantify the hydrophobic nature of proteins and protein domains. It may aid in evaluating changes associated with post-translational protein modification and protein-protein interactions in a defined system.
Highlights
§ Present address: Pulmonary and Critical Care Medicine, Decatur VA Medical Center, Emory University, VAMC Rm. 1253, 1670 Clairmont Rd., Decatur, GA 30033
The carboxyl terminus is important in the membrane association of Gsa [17, 18], and the carboxyl terminus of Gta was postulated to be involved in anchoring the Gta·phosphodiesterase complex to membranes [19]
The data are consistent with a role for a myristoylated amino terminus in membrane association, with the more recent recognition that several G" subunits are palmitoylated, the contribution of the less hydrophobic myristate may need to be
Summary
§ Present address: Pulmonary and Critical Care Medicine, Decatur VA Medical Center, Emory University, VAMC Rm. 1253, 1670 Clairmont Rd., Decatur, GA 30033. Membrane association may occur directly via the myristoyl moiety and its neighboring amino acids, as has been postulated for the Src protein [29, 30], or indirectly if it increases the association of the a subunit with the prenylated and membrane-associated G13'Y subunits [31, 32] In support of the latter hypothesis, both This partitioning difference has been the basis for separating integral from peripheral membrane proteins [34] We investigated this partitioning procedure as a new way of characterizing the hydrophobic and membrane-association domains of native and recombinant Gta and myristoylated and nonmyristoylated rGoa as well as possibly assessing protein-protein interactions in a purified defined system
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