Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

Abstract

RGS4, a mammalian GTPase activating protein for G protein ␣ subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae. To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system. Deletion of the N-terminal 33 aa of RGS4 (⌬1-33) yielded a nonfunctional protein and loss of plasma membrane localization. These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (⌬1-33). Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to green f luorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain. RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation. Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling. Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.Heterotrimeric G proteins couple receptors for hormones, neurotransmitters, and sensory signals to intracellular effector molecules, thereby eliciting cellular responses (1, 2). Guanine nucleotide exchange and hydrolysis on the G protein ␣ subunit drives the cycle of activation and deactivation of these signaling pathways. The duration of G protein-mediated responses is subject to the intrinsic GTPase rate of the G protein ␣ subunit, but is also modulated by extrinsic factors. A recently appreciated form of regulation has come from the discovery that members of a protein family called regulators of G protein signaling, or RGS proteins, stimulate the rate of GTP hydrolysis by G protein ␣ subunits (3-5). RGS proteins are found in species ranging from yeast to mammals and constitute a family of at least 20 mammalian proteins (6-8).All RGS family members share sequence similarity that extends over approximately 130 aa, separated in some cases by insertions of varying length (9-11). This conserved RGS domain is sufficient to stimulate GTPase activity of G protein ␣ subunits in vitro (12)(13)(14). Expression of the RGS homology domain of RET-RGS1 or RGS4 yields a recombinant protein that is a functional GAP (GTPase-activating protein). In the crystal structure of RGS4 bound to AlF 4 Ϫ -activated G i␣1 , only the core domain is visible (15). The RGS homology domain binds to the switch regions of G i␣1 and appears to catalyze GTP hydrolysis by stabilizing the switch regions of the G protein in a conformation that favors the transition state of the reactants (14, 15). Sequences outside the RGS homology domain exhibit considerable diversity among family members.Although rapid progress has been made in dissecting the biochemical mechanism by which RGS proteins regulate G protein a...

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

“…When viewed by fluorescence microscopy, GFP-CTSte18 was localized to the plasma membrane ( Figure 4B), confirming that the last nine amino acids of Ste18p (Table 2) are sufficient to target a heterologous protein to the plasma membrane (Srinivasa et al, 1998). Mutation of the cysteine corresponding to C106 in Ste18p, the thioacylation site, or the residue corresponding to the farnesylation site (equivalent to C107) resulted in a loss of plasma membrane localization, suggesting that both lipid modifications are required for plasma membrane association in the absence of protein-protein interactions with Gpa1p ( Figure 4B).…”

α-Synuclein disrupts stress signaling by inhibiting polo-like kinase Cdc5/Plk2

“…To determine whether αS toxicity is due to membrane binding, two different C-terminal membrane-targeting sequences (mts) that should increase the amount αS on the plasma membrane were tested. Mts1 (SNSVCCTLM) and mts2 (GSGGCCLLS) are derived from the yeast proteins Ste18 and Ras2, respectively; in vivo, these tags are palmitoylated, and the resultant palmitoylated protein localizes to the plasma membrane (16). Cells expressing untagged αS exhibited impaired growth compared with vector control cells, whereas cells expressing αS-mts1 or αS-mts2 exhibited an even more pronounced growth defect than cells expressing the untagged protein (Fig.…”

RGS4 Is Arginylated and Degraded by the N-end Rule Pathway in Vitro

“…8) Similarities among the N-terminal sequences of RGS4, RGS5, and RGS16 (67) suggested that RGS5 and RGS16 may also be N-end rule substrates. This prediction was tested, thus far, with RGS16, and was confirmed.…”

“…The encoded N-terminal sequence of RGS4 is similar to those of RGS5 and RGS16; the similarities include Cys-2 and a basic residue at position 3 (55,67). To test whether RGS16 was an N-end rule substrate, we expressed RGS16 in reticulocyte lysate.…”

“…An analogous protection of RGS4 against targeting by the N-end rule pathway in L cells might involve a much higher fraction of the newly formed RGS4. The mechanism of protection may be a modification of the N-terminal Cys, for example, its palmitoylation (45,47,62,67) or acetylation (68) that would be expected to preclude the arginylation and degradation of RGS4 by the N-end rule pathway. Thus it is possible, indeed likely, that there is a kinetic competition among these reactions at the N-terminal Cys of a nascent RGS4 protein.…”