Plasma membrane localization of G alpha z requires two signals

Abstract

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the alpha subunits are myristoylated and/or palmitoylated, whereas the gamma chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled alpha subunit, alpha z. Wild-type alpha z (alpha z-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does alpha z-WT. Removal of the myristoylation site produced a mutant alpha z that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of beta gamma with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on alpha z-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on alpha z-WT. Together these results suggest that myristate and beta gamma promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on alpha z specific localization at the plasma membrane.

Figure 1

Activation of HA-MAPK by α_z and mutants. cDNAs encoding D₂R and either α_z-WT, α_z-G2A, α_z-C3A, α_z-G2AC3A, or vector were transfected into CHO-K1 cells. Cells were serum starved for 24 h. After 4 h of pretreatment with or without PTX, cells were treated with serum-free medium (basal) or 10 μM quinpirole for 7 min. HA-MAPK activity was measured as described in MATERIALS AND METHODS. Bars, mean ± 2 SE of triplicate determinations. Similar results were obtained in two additional experiments.

Figure 2

Particulate versus soluble distribution of α_z mutants with or without βγ. CHO-K1 cells were transiently transfected with α_z mutants alone (A) or transfected with α_z mutants plus β₁ and γ₂ (B). Crude particulate (P) and soluble (S) fractions were prepared as described in MATERIALS AND METHODS. Equivalent proportions of each fraction were analyzed by SDS-PAGE and Western blotting with the EE mAb.

Figure 3

Incorporation of palmitate by α_z mutants. CHO-K1 cells, transiently transfected with α_z mutants, were incubated with 0.75 mCi/ml [³H]palmitic acid for 2 h. α_z was immunoprecipitated from total cell extracts with the EE mAb followed by SDS-PAGE. Identical gels were treated with either 1 M Tris (pH 7.0) or 1 M hydroxylamine (pH 7.0) for 12 h. Gels were then processed for fluorography (19-d exposure).

Figure 4

Subcellular localization of WT and mutant α_z in stably transfected cells. CHO-K1 cells were stably transfected with pcDNA3 vector alone (A), α_z-WT (B), α_z-G2A (C), α_z-C3A (D), or α_z-G2AC3A (E). Cells were plated onto glass coverslips and fixed with 4% formaldehyde in PBS. Permeabilized cells were incubated with EE mAb followed by incubation with FITC-conjugated antimouse antibody. Images were obtained by confocal laser scanning microscopy. Bar, 10 μm

Figure 5

Immunofluorescence pattern of α_z following SLO treatment. CHO-K1 cell lines stably expressing α_z-WT (A and B), α_z-G2A (C–F), or α_z-C3A (G and H) were treated with SLO as described in MATERIALS AND METHODS and fixed. α_z was detected by incubation with EE mAb followed by FITC-conjugated antimouse antibody (A, C, E, and G). The same cells were stained with Texas Red-conjugated phalloidin to visualize actin (B, D, and H) or with rabbit anti-mannosidase II followed by Texas Red-conjugated antirabbit antibodies to visualize Golgi (F). Images were obtained by confocal laser scanning microscopy. Bar, 10 μm

Figure 6

Effect of coexpressing βγ on activation of HA-MAPK by α_z-G2A and α_z-G2AC3A. D₂R was transiently transfected into CHO-K1 cells with vector, α_z-G2A, α_z-G2A plus β₁ and γ₂, α_z-G2AC3A, or α_z-G2AC3A plus β₁ and γ_2. Cells were serum starved for 24 h and pretreated with PTX for 4 h before stimulating with serum-free medium (basal) or 10 μM quinpirole for 7 min. HA-MAPK activity was assayed as described in MATERIALS AND METHODS. Bars, mean ± 2 SE of triplicate determinations. Similar results were obtained in two separate experiments.

Figure 7

Effect of βγ on palmitate incorporation by α_z-G2A and the cellular distribution of palmitoylated α_z-G2A. (A) α_z-WT and α_z-G2A were separately transfected into CHO-K1 cells without or with β₁ and γ₂. Palmitate incorporation was determined as described in the legend of Figure 3. A third identical gel was analyzed by Western blotting with the EE mAb. (B) CHO-K1 cells transfected with α_z-G2A, β₁ and γ₂ were labeled with [³H]palmitate for 2 h. Following radiolabeling, particulate (P) and soluble (S) extracts were prepared. After immunoprecipitation with the EE mAb, proteins were resolved by SDS-PAGE and visualized by fluorography (21-d exposure); an identical gel was analyzed by Western blotting with the EE mAb.

Figure 8

Effect of βγ on the subcellular localization of α_z-G2A. CHO-K1 cells transiently transfected with α_z-G2A (A, C, and D) or α_z-G2A plus β₁ and γ₂ (B, E, and F) were fixed after no treatment (A and B) or after treatment with SLO (C–F). α_z-G2A was detected by incubation with the EE mAb followed by incubation with FITC-conjugated antimouse antibody (A–C and E). Texas Red-conjugated phalloidin-stained actin filaments (D and F). Bar, 10 μm

Figure 9

Subcellular localization of α_z and βγ. CHO-K1 cells transiently transfected with β₁ and γ₂ plus α_z-WT (A and B) or α_z-G2A (C and D) were fixed in 4% formaldehyde. α_z was detected by incubation with EE mAb followed by FITC-conjugated antimouse antibody (A and C). γ₂ was detected by incubation with polyclonal antiserum against γ₂ followed by Texas Red antirabbit antibody (B and D). Bar, 10 μm.

Figure 10

Turnover of palmitate on α_z-WT and α_z-G2A plus βγ. Cells were transfected with either D₂R and α_z-WT (A) or D₂R, α_z-G2A, β₁, and γ₂ (B). Cells were incubated with 0.75 mCi/ml [³H]palmitate for 2 h. After radiolabeling cells were incubated in chase medium in the presence or absence of 10 μM quinpirole. At indicated times, cells (100-mm plate) were harvested and α_z was immunoprecipitated from the total cell extract. Depalmitoylation was determined by densitometry of the fluorographs (A, 12-d exposure; B, 9-d exposure).