11 - Purification and Properties of a GTPase-Activating Protein for Yeast Rab GTPases
Introduction
Rab proteins constitute the largest family of small GTP binding proteins.1 A total of 11 genes encoding Rab proteins is found in the yeast genome. Among them, Sec4p was the first to be implicated in vesicle traffic.2 When the function of Sec4p is blocked, cells accumulate Golgi-derived secretory vesicles.
GTP binding proteins cycle between the GTP-bound active form and the GDP-bound inactive form. The conversion between these two forms is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Yeast proteins Gyp6p and Gyp7p were the first Rab GAPs to be cloned,3,4 but they are not active on Sec4p. GAP activity for Sec4p was first observed in yeast cell lysate.5 To clone a Sec4p-specific GAP, we searched the sequence database and found an uncharacterized yeast open reading frame that shares homology with Gyp6p and Gyp7p. We named this protein Gyp1p and demonstrated that it is a GAP for Sec4p.6 Independently, a more systematic analysis of the homology shared by yeast Rab GAPs has been published.7 The homologous domain defined in that paper has been shown to be the GAP catalytic domain in Gyp1p and Gyp7p.8 This domain is not limited to yeast proteins or to Rab GAPs. A human protein called CAPCenA is a Rab6 GAP and shares this domain.9 Interestingly, one Schizosaccharomyces pombe protein sharing this homology, Cdc16, has been shown to be one subunit of a heterodimeric GAP for a GTPase involved in cell cycle regulation.10
In this chapter, we present the methods that we used in the study of Gyp1p.
Section snippets
Construction of Expression Plasmids
Polymerase chain reaction (PCR) was used to amplify the open reading frames of Gyp1p, Ypt1p, Ypt6p, Ypt7p, Ypt32p, and Ypt51p (Research Genetics, Huntsville, AL) with the following primer pair:
Forward primer: 5′-CGGGATCCACTCGAGCATATGGAATTCCAGCTGACCACC-3′
Back primer: 5′-GGAATTCCATATGCTCGAGGATCCCCGGGAATTGCCATG-3′
The PCR products were digested with NdeI and BamHI, and then cloned into the expression vector pET15b (Novagen, Madison, WI) so that each open reading frame is fused in frame to the
Solutions
Buffer stock: 10× sonication and wash buffer stock, 0.5 M sodium phosphate, pH 8.0 or 6.0, 3 M NaCl; 5 M imidazole, adjust to pH 8.0. Make the following buffers just before use:
Sonication buffer: 50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 5 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1% Tween 20
Wash buffer: 50 mM sodium phosphate, pH 6.0, 0.3 M NaCl, 10% glycerol, 5 mM 2-mercaptoethanol
2× column buffer: 40 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 10 mM 2-mercaptoethanol,
Reagents
35S-labeled GTPγS (NEN, Boston, MA), 12.5 mCi/ml in 10 mM Tricine, pH 7.6, 10 mM DTT: The specificity is 1000 Ci/mmol, so the actual concentration of GTPγS is 12.5 μM. The size that we order is 250 μCi, i.e., 20 μl volume. It can be diluted fivefold with 50 mM Tris-HCl, pH 8.0, 10 mM DTT, and aliquoted to 5 tubes and frozen at −80°. The scintillation counting efficiency of 35S is almost 100%, so 1 μCi corresponds to 2 × 106 cpm. The activity of the original material would be 2.5 × 107 cpm/μl,
Reagents
[α-32P]GTP (NEN): 3000 Ci/mmol, 10 mCi/ml in 50 mM Tricine (pH 7.6). The actual GTP concentration is about 3.33 μM. Make a stock solution: Dilute the [α-32P]GTP 10 times in 10 μM GTP, 50 mM Tris-HCl, pH 8.0, 1 mM DTT, and 1 mM ATP.
Polyethyleneimine-cellulose thin-layer chromatography (TLC) plates with fluorescent indicator (Fisher Scientific, Springfield, NJ): Predevelop in distilled water and dry before use.
10× buffer A: 500 mM Tris-HCl, pH 8.0, 10 mg/ml BSA, 10 mM DTT, 10 mM ATP.
Preloading GTPase
Incubate 2 μM
Reagents
[γ-32P]GTP (NEN): 6000 Ci/mmol, 10 mCi/ml in 50 mM Tricine (pH 7.6), stored at 4°. The actual GTP concentration is about 1.67 μM. Make a stock solution: 10 μM GTP in 50 mM Tris-HCl, pH 8.0, 1 mM DTT, and 1 mM ATP. Add [γ-32 P]GTP so the final activity is about 4 × 105 cpm/μl (1/50 volume of [γ-32 P]GTP, if it is fresh).
10× buffer B: 500 mM sodium N-2 hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-NaOH, pH 6.8, 10 mg/ml BSA, 10 mM DTT, and 10 mM ATP. (The optimal pH for Gyp1p catalyzed
Summary of Results
The methods presented here have been used to characterize Gyp1p.6 We found that Gyp1p stimulates the hydrolysis of Sec4p-bound GTP to GDP, but Gyp1p itself does not have GTPase activity. Gyp1p can act together with the GEF of Sec4p, Sec2p, to enhance the steady- state rate of GTP hydrolysis by Sec4p. Under single turnover conditions, the accelerated rate of GTP hydrolysis is directly proportional to the concentration of Gyp1p, and the maximal enhancement is more than 200-fold, all indicating
Acknowledgment
This work was supported by grants from the National Institutes of Health to P.N.
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