Membrane targeting of Rab GTPases is influenced by the prenylation motif

Abstract

Rab GTPases are regulators of membrane traffic. Rabs specifically associate with target membranes via the attachment of (usually) two geranylgeranyl groups in a reaction involving Rab escort protein and Rab geranylgeranyl transferase. In contrast, related GTPases are singly prenylated by CAAX prenyl transferases. We report that di-geranylgeranyl modification is important for targeting of Rab5a and Rab27a to endosomes and melanosomes, respectively. Transient expression of EGFP-Rab5 mutants containing two prenylatable cysteines (CGC, CC, CCQNI, and CCA) in HeLa cells did not affect endosomal targeting or function, whereas mono-cysteine mutants (CSLG, CVLL, or CVIM) were mistargeted to the endoplasmic reticulum (ER) and were nonfunctional. Similarly, Rab27aCVLL mutant is also mistargeted to the ER and transgenic expression on a Rab27a null background (Rab27aash) did not rescue the coat color phenotype, suggesting that Rab27aCVLL is not functional in vivo. CAAX prenyl transferase inhibition and temperature-shift experiments further suggest that Rabs, singly or doubly modified are recruited to membranes via a Rab escort protein/Rab geranylgeranyl transferase-dependent mechanism that is distinct from the insertion of CAAX-containing GTPases. Finally, we show that both singly and doubly modified Rabs are extracted from membranes by RabGDIalpha and propose that the mistargeting of Rabs to the ER results from loss of targeting information.

Figure 1.

In vitro prenylation of recombinant Rab5a proteins by protein prenyl transferases. (A) Approximately 2 μg of each recombinant small GTPase was subjected to SDS-PAGE and visualized by staining with Coomassie-Blue. (B) RGGT assay. Each reaction contained 25 nM RGGT, 5 μM [³H]GGPP (3000 dpm/pmol), 10 μM either Rab5aCCSN (•), Rab5aCSLG (•), Rab5aCVLL (○), Rab5aCVIM (•), or Rab5aSSSN (▪), and the indicated concentrations of REP. (C) PGGT assay. Each reaction contained 20 nM PGGT, 5 μM [³H]GGPP (3000 dpm/pmol), and the indicated concentrations of Rac1 (•), Rab5aCCSN (○), Rab5aCSLG (▪), Rab5aCVLL (•), Rab5aCVIM (•), or Rab5aSSSN (□). (D) PFT assay. Each reaction contained 40 nM recombinant PFT, 1.2 μM [³H]FPP (11,275 dpm/pmol), and the indicated concentrations of K-Ras (•), Rab5aCCSN (○), Rab5aCSLG (▪), Rab5aCVLL (•), Rab5aCVIM (•), or Rab5aSSSN (□). After 37°C incubation for 10 min (RGGT and PGGT assays) or 15 min (PFT assay), the amount of [³H]GGPP or [³H]FPP transferred to each protein was determined as described under MATERIALS AND METHODS. Each value is the average of triplicate determinations.

Figure 2.

Transient overexpression of EGFP-Rab5a wild-type and double-cysteine mutant proteins in HeLa cells. HeLa cells were transfected with EGFP-Rab5a (A–C), EGFP-Rab5aCGC (D–F), EGFP-Rab5aCCQNI (G–I), or with the respective constitutively active constructs, EGFP-Rab5aQ79L (J–L), EGFP-Rab5aQ79L, CGC (M–O) and EGFP-Rab5aQ79L, CCQNI (P–R). After 24 h, cells were permeabilized, fixed, and immunostained with an anti-TfR antibody (A–I) or with anti-EEA1 antibody (Panels J-R) as described under MATERIALS AND METHODS. The panels on the right show the merged fluorescent signals. Bar, 20 μm.

Figure 2.

Transient overexpression of EGFP-Rab5a wild-type and double-cysteine mutant proteins in HeLa cells. HeLa cells were transfected with EGFP-Rab5a (A–C), EGFP-Rab5aCGC (D–F), EGFP-Rab5aCCQNI (G–I), or with the respective constitutively active constructs, EGFP-Rab5aQ79L (J–L), EGFP-Rab5aQ79L, CGC (M–O) and EGFP-Rab5aQ79L, CCQNI (P–R). After 24 h, cells were permeabilized, fixed, and immunostained with an anti-TfR antibody (A–I) or with anti-EEA1 antibody (Panels J-R) as described under MATERIALS AND METHODS. The panels on the right show the merged fluorescent signals. Bar, 20 μm.

Figure 3.

Transient overexpression of EGFP-Rab5a single-cysteine mutant proteins in HeLa cells. HeLa cells were transfected with EGFP-Rab5aCSLG (A–C), EGFP-Rab5aCVLL (D–F), EGFP-Rab5aCVIM (G–I); with the respective constitutively active constructs, EGFP-Rab5aQ79L, CSLG (M–O), EGFP-Rab5aQ79L, CVLL (P–R), and EGFP-Rab5aQ79L, CVIM (S–U), or cotransfected with EGFP-Rab5a and myc-Rab5aCVLL (our unpublished data) as well as EGFP-Rab5aCSLG and myc-Rab5aCVLL (J–L). After 24 h, cells were permeabilized, fixed, and immunostained with an anti-TfR, anti-EEA1, and anti-myc antibodies (middle) as indicated and as described under MATERIALS AND METHODS. The panels on the right show the merged fluorescent signals. Bar, 20 μm.

Figure 3.

Transient overexpression of EGFP-Rab5a single-cysteine mutant proteins in HeLa cells. HeLa cells were transfected with EGFP-Rab5aCSLG (A–C), EGFP-Rab5aCVLL (D–F), EGFP-Rab5aCVIM (G–I); with the respective constitutively active constructs, EGFP-Rab5aQ79L, CSLG (M–O), EGFP-Rab5aQ79L, CVLL (P–R), and EGFP-Rab5aQ79L, CVIM (S–U), or cotransfected with EGFP-Rab5a and myc-Rab5aCVLL (our unpublished data) as well as EGFP-Rab5aCSLG and myc-Rab5aCVLL (J–L). After 24 h, cells were permeabilized, fixed, and immunostained with an anti-TfR, anti-EEA1, and anti-myc antibodies (middle) as indicated and as described under MATERIALS AND METHODS. The panels on the right show the merged fluorescent signals. Bar, 20 μm.

Figure 4.

Transient overexpression of EGFP-Rab27a wild-type and mutant in cultured melanocytes. melan-c cells were transfected with EGFP-Rab27a (A–F) or EGFP-Rab27aCVLL (G–L). After 48 h, cells were permeabilized, fixed, and immunostained with either an anti-TRP1 antibody (B and H) or with an anti-calnexin antibody (E and K) as described under MATERIALS AND METHODS. The panels on the right show the merged fluorescent signals. Bar, 20 μm.

Figure 5.

Transgenic expression of Rab27a and Rab27aCVLL in ashen mice. (A) Organization of transgenic construct encoding Rab27aCVLL under the control of β-actin promoter. The P_CAG/mycRab27/β-globin construct contains the CMV enhancer (dark green) and the chicken β-actin promoter sequence (light green) upstream of a myc-tag (dark blue) in frame with Rab27a cDNA (light blue), followed by the rabbit β-globin poly(A) sequence (red). (B and C) Photography of representative mice for a Rab27aCVLL rescue experiment. Littermates resulting from a cross between a homozygous ashen mouse with a heterozygous transgenic mouse were genotyped as described under MATERIALS AND METHODS. In B from left to right are shown an ashen homozygous mouse that does not carry the transgene (ash/ash), an ashen homozygous mouse expressing transgenic Rab27aCVLL (ash/ash, —/tg^Rab27aCVLL), and a heterozygous control mouse (+/ash). In C from left to right are shown an ashen homozygous mouse expressing transgenic Rab27a (ash/ash, —/tg^Rab27a), a heterozygous control mouse (+/ash), and ashen homozygous mouse that does not carry the transgene (ash/ash). (D–F) Primary melanocytes from littermate ash/ash (D), ash/ash, —/tg^Rab27CVLL (E), or wild type +/+ (F) were cultured and subjected to phase contrast light microscopy as described under MATERIALS AND METHODS. Bars, 20 μm.

Figure 6.

Intracellular localization of mono-cysteine Rab5a mutant proteins after transient transfection of HeLa cells. HeLa cells were transfected with either EGFP-CVLL (A–C), EGFP-CVLL and myc-Rab5aCVLL (D–F), EGFP-CVIM, and myc-Rab5aCVLL (G–I), EGFP-CSLG and myc-Rab5aCVLL (J–L), or EGFP-CSLG (M–X). After 24 h, cells were washed, permeabilized, fixed, and immunostained with either an anti-calnexin antibody (A–C and M–O), an anti-myc antibody (D–L), an anti-ERGIC-53 antibody (P–R), or an anti-Golgin-97 antibody (S–X) as described under MATERIALS AND METHODS. The cells depicted in V–X were treated for 1 h with brefeldin-A as described under MATERIALS AND METHODS. The panels on the right show the merged fluorescent signals. Bar, 20 μm.

Figure 6.

Intracellular localization of mono-cysteine Rab5a mutant proteins after transient transfection of HeLa cells. HeLa cells were transfected with either EGFP-CVLL (A–C), EGFP-CVLL and myc-Rab5aCVLL (D–F), EGFP-CVIM, and myc-Rab5aCVLL (G–I), EGFP-CSLG and myc-Rab5aCVLL (J–L), or EGFP-CSLG (M–X). After 24 h, cells were washed, permeabilized, fixed, and immunostained with either an anti-calnexin antibody (A–C and M–O), an anti-myc antibody (D–L), an anti-ERGIC-53 antibody (P–R), or an anti-Golgin-97 antibody (S–X) as described under MATERIALS AND METHODS. The cells depicted in V–X were treated for 1 h with brefeldin-A as described under MATERIALS AND METHODS. The panels on the right show the merged fluorescent signals. Bar, 20 μm.

Figure 7.

Effect of the PGGT inhibitor GGTI-298 on membrane targeting of small GTPases. HeLa cells were transiently transfected with EGFP-Cdc42 (A and B), EGFP-CVLL (C and D), EGFP-Rab5aCVLL (E and F), EGFP-Rab5aCSLG (G and H), EGFP-Rab5a (I and J), or EGFP-HRas (K and L) for 4 h, followed by incubation for 20 h in the absence or presence of 15 μM GGTI-298 as indicated. Cells were then permeabilized, fixed and visualized as described under MATERIALS AND METHODS. Bar, 20 μm.

Figure 8.

Effect of temperature shift on membrane targeting of small GTPases. HeLa cells were transfected with either EGFP-HRas (A–C), EGFP-Rab5a (D–F), or EGFP-Rab5aCVLL (G–I) for 4 h and subjected to one of the indicated treatments: 3 h at 20°C (A, D, and G); 3 h at 20°C followed by 1 h at 37°C (B, E, and H); or 3 h at 37°C (C, F, and I). Subsequently, cells were permeabilized, fixed, and visualized as described under MATERIALS AND METHODS. Bar, 20 μm.

Figure 9.

GDI extraction of Rab5a wild-type and mono-cysteine mutants. HEK293 cells transiently transfected with EGFP-Rab5a, EGFP-Rab5aCVLL, or EGFP-Rab5aCSLG were homogenized, fractionated into membrane and soluble fractions, and the membrane fraction (denoted Total) was subjected to extraction with the indicated concentrations of GST-GDIα in the presence or absence of GDP or GTPγS as indicated, followed by SDS-PAGE as described under MATERIALS AND METHODS. The EGFP-Rab5a proteins were visualized by immunoblotting with an anti-Rab5a antibody. Calnexin was used as a loading control (our unpublished data).