Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane.

The GPI (glycosylphosphatidylinositol) moiety is attached to newly synthesized proteins in the lumen of the ER (endoplasmic reticulum). The modified proteins are then directed to the PM (plasma membrane). Less well understood is how nascent mammalian GPI-anchored proteins are targeted from the ER to the PM. In the present study, we investigated mechanisms underlying membrane trafficking of the GPI-anchored proteins, focusing on the early secretory pathway. We first established a cell line that stably expresses inducible temperature-sensitive GPI-fused proteins as a reporter and examined roles of transport-vesicle constituents called p24 proteins in the traffic of the GPI-anchored proteins. We selectively suppressed one of the p24 proteins, namely p23, employing RNAi (RNA interference) techniques. The suppression resulted in pronounced delays of PM expression of the GPI-fused reporter proteins. Furthermore, maturation of DAF (decay-accelerating factor), one of the GPI-anchored proteins in mammals, was slowed by the suppression of p23, indicating delayed trafficking of DAF from the ER to the Golgi. Trafficking of non-GPI-linked cargo proteins was barely affected by p23 knockdown. This is the first to demonstrate direct evidence for the transport of mammalian GPI-anchored proteins being mediated by p24 proteins.

Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling.

The G protein subunit, betagamma, plays an important role in targeting alpha subunits to the plasma membrane and is essential for binding and activation of the heterotrimer by heptahelical receptors. Mutation of residues in the N-terminal alpha-helix of alpha s and alpha q that contact betagamma in the crystal structure of alpha i reduces binding between alpha and betagamma, inhibits plasma membrane targeting and palmitoylation of the alpha subunit, and results in G proteins that fail to couple receptor activation to stimulation of effector. Overexpression of betagamma can recover this loss of signaling through Gs but not Gq. In fact, a single mutation (I25A) in alpha q can block alpha q-mediated generation of inositol phosphates. Function is not recovered by betagamma overexpression nor myristoylation directed plasma membrane localization. Introduction of a Q209L activating mutation with I25A results in a constitutively active alpha q as expected, but surprisingly a R183C activating mutation does not result in constitutive activity when present with I25A. Examination of binding between alpha and betagamma via a pull down assay shows that the N-terminal betagamma-binding mutations inhibit alpha-betagamma binding significantly more than the R183C or Q209L activating mutations do. Moreover, introduction of the I25A mutation into alpha q RC disrupts co-immunoprecipitation with PLCbeta1. Taken together, results presented here suggest that alpha-betagamma binding is necessary at a point downstream from receptor activation of the heterotrimeric G protein for signal transduction by alpha q.

Palmitoylation and plasma membrane targeting of RGS7 are promoted by alpha o.

Regulator of G protein signaling (RGS) proteins modulate G protein signaling by acting as GTPase-activating proteins for G protein alpha-subunits. RGS7 belongs to a subfamily of RGS proteins that exist as dimers with the G protein beta(5)-subunit. In this report, we addressed the mechanisms of plasma membrane localization of beta(5)RGS7. When expressed in human embryonic kidney 293 cells, beta(5)RGS7 was found to be cytoplasmic and soluble. Expression of alpha(o) promoted a strong redistribution of beta(5)RGS7 to the plasma membrane. Expression of alpha(q), however, failed to affect the subcellular localization of beta(5)RGS7. The constitutively active mutant alpha(o)R179C, like wild-type alpha(o), strongly recruited beta(5)RGS7 to plasma membranes; however, inactive alpha(o)G204A, RGS-insensitive alpha(o)G184S, and lipidation-deficient alpha(o)G2A were all defective in the ability to promote plasma membrane localization of beta(5)RGS7. In addition, palmitoylation of RGS7 was demonstrated, and palmitoylation required expression of alpha(o) or alpha(o)R179C. To examine potential palmitoylation sites of RGS7, several cysteines were substituted with serines. beta(5)RGS7C133S failed to localize to plasma membranes when coexpressed with alpha(o), suggesting cysteine 133 of RGS7 as a putative palmitoylation site. Finally, deletion of amino acids 76 to 128 of RGS7, which includes part of the disheveled, EGL-10, pleckstrin (DEP) domain, prevented alpha(o)-mediated plasma membrane recruitment of beta(5)RGS7. These findings are the first to demonstrate Galpha-regulated plasma membrane localization and palmitoylation of beta(5)RGS7 and suggest that membrane targeting of beta(5)RGS7 is a complex process requiring at least RGS domain-mediated interaction with alpha(o) and RGS7 palmitoylation.

Exocytic pathway-independent plasma membrane targeting of heterotrimeric G proteins.

Heterotrimeric G proteins are lipid-modified, peripheral membrane proteins that function at the inner surface of the plasma membrane (PM) to relay signals from cell-surface receptors to downstream effectors. Cellular trafficking pathways that direct nascent G proteins to the PM are poorly defined. In this report, we test the proposal that G proteins utilize the classical exocytic pathway for PM targeting. PM localization of the G protein heterotrimers alpha s beta 1 gamma 2 and alpha q beta 1 gamma 2 occurred independently of treatment of cells with Brefeldin A, which disrupts the Golgi, or expression of Sar1 mutants, which prevent the formation of endoplasmic reticulum to Golgi transport vesicles. Moreover, the palmitoylation of alpha q was unaffected by Brefeldin A treatment, even though the palmitoylation of SNAP25 was blocked by Brefeldin A. Non-palmitoylated mutants of alpha s and alpha q failed to stably bind to beta gamma and displayed a dispersed cytoplasmic localization when co-expressed with beta gamma. These findings support a refined model of the PM trafficking pathway of G proteins, involving assembly of the heterotrimer at the endoplasmic reticulum and transport to the PM independently of the Golgi.

Heterotrimer formation, together with isoprenylation, is required for plasma membrane targeting of Gbetagamma.

Nascent beta and gamma subunits of heterotrimeric G proteins need to be targeted to the cytoplasmic face of the plasma membrane (PM) in order to transmit signals. We show that beta(1)gamma(2) is poorly targeted to the PM and predominantly localized to endoplasmic reticulum (ER) membranes when expressed in HEK293 cells, but co-expression of a G protein alpha subunit allows strong PM localization of the beta(1)gamma(2). Furthermore, C-terminal isoprenylation of the gamma subunit is necessary but not sufficient for PM localization of beta(1)gamma(2). Isoprenylation of gamma(2) and localization of beta(1)gamma(2) to the ER occurs independently of alpha expression. Efficient PM localization of beta(1)gamma(2) in the absence of co-expressed alpha is observed when a site for palmitoylation, a putative second membrane targeting signal, is introduced into gamma(2). When a mutant of alpha(s) is targeted to mitochondria, beta(1)gamma(2) follows, consistent with an important role for alpha in promoting subcellular localization of betagamma. Furthermore, we directly demonstrate the requirement for alpha by showing that disruption of heterotrimer formation by the introduction of alpha binding mutations into beta(1) impedes PM targeting of beta(1)gamma(2). The results indicate that two membrane targeting signals, lipid modification and alpha binding, make concerted contributions to PM localization of betagamma.