Hydrophobicity and Subunit Interactions of Rod Outer Segment Proteins Investigated Using Triton X-114 Phase Partitioning

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 G , partitioned into the detergent-rich phase. Arrestin, a soluble protein, accumulated in the aqueous phase. G distributed about equally between phases whether GDP (G •GDP) or GTP (G •GTP) was bound. G increased recovery of G •GDP but not G •GTP in the detergent phase. Trypsin-treated G , which lacks the fatty acylated amino-terminal 2-kDa region, accumulated to a greater extent in the aqueous phase than did intact G . Trypsinized cGMP phosphodiesterase, which lacks the isoprenyl group, partitioned into the aqueous phase. A carboxyl-terminal truncated mutant (Val-331 stop) of G accumulated more in the aqueous phase then did recombinant full-length G , supporting the role of the carboxyl terminus in increasing its hydrophobicity. N -Myristoylated recombinant G was more hydrophobic than recombinant G without myristate. ADP-ribosylation of G 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.