Nascent ␤ and ␥ 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 ␤ 1 ␥ 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 ␣ subunit allows strong PM localization of the ␤ 1 ␥ 2 . Furthermore, C-terminal isoprenylation of the ␥ subunit is necessary but not sufficient for PM localization of ␤ 1 ␥ 2 . Isoprenylation of ␥ 2 and localization of ␤ 1 ␥ 2 to the ER occurs independently of ␣ expression. Efficient PM localization of ␤ 1 ␥ 2 in the absence of co-expressed ␣ is observed when a site for palmitoylation, a putative second membrane targeting signal, is introduced into ␥ 2 . When a mutant of ␣ s is targeted to mitochondria, ␤ 1 ␥ 2 follows, consistent with an important role for ␣ in promoting subcellular localization of ␤␥. Heterotrimeric G proteins 1 are composed of ␣ and ␤␥ subunits. The ␤␥ complex only dissociates when denatured and hence is a functional monomer under physiological conditions. Upon receptor activation the ␤␥ dimer is freed from GTP-bound ␣ and relays signals to downstream molecules until it reassociates with GDP-bound ␣, re-forming the heterotrimer. To perpetuate this G protein cycle, the trimer must be tethered to the cytoplasmic face of the PM. This crucial subcellular localization is promoted by the covalent attachment of lipids to the subunits. Three lipid modifications have been found in G proteins, namely myristoylation and/or palmitoylation for the ␣ subunit and isoprenylation for the ␥ subunit. Myristoylation is the covalent attachment of a 14-carbon saturated myristate to an N-terminal glycine through an amide bond, whereas palmitoylation is a 16-carbon saturated palmitate linked to a cysteine via a thioester bond. Isoprenylation is a lipid modification in which an unsaturated, 15-carbon farnesyl isoprenoid or 20-carbon geranylgeranyl isoprenoid is linked to a cysteine, via a thioether bond.Mechanisms underlying the PM targeting of the ␣ subunit have been studied in some detail (1-3). The available data suggest a model in which myristoylation and/or binding to ␤␥ subunits constitutes an initial membrane targeting signal for the ␣ subunit. Subsequently, palmitoylation functions as a second signal that specifies localization to the PM. In contrast to the ␣ subunit, relatively less is known about how ␤␥ is targeted to the PM. Either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is linked to a cysteine residue in the so-called CAAX motif in the C terminus of the ␥ subunit (4, 5). The CAAX box (where C is a cysteine, A is commonly an aliphatic amino acid, and X can be one of several amino acids) is a consensus sequence for isoprenylation. The X residue is thought to specify which isoprenoid group will be linked to the cysteine. Among 12 human ␥ subunits thus far identified, ␥ 1 , ␥ 9 , and ␥ 11 have serine in the X position and are farnesylated, and the rest of ...

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.

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