Structure-function analysis of the yeast mitochondrial Rho GTPase, Gem1p: implications for mitochondrial inheritance

Abstract

Mitochondria undergo continuous cycles of homotypic fusion and fission, which play an important role in controlling organelle morphology, copy number, and mitochondrial DNA maintenance. Because mitochondria cannot be generated de novo, the motility and distribution of these organelles are essential for their inheritance by daughter cells during division. Mitochondrial Rho (Miro) GTPases are outer mitochondrial membrane proteins with two GTPase domains and two EF-hand motifs, which act as receptors to regulate mitochondrial motility and inheritance. Here we report that although all of these domains are biochemically active, only the GTPase domains are required for the mitochondrial inheritance function of Gem1p (the yeast Miro ortholog). Mutations in either of the Gem1p GTPase domains completely abrogated mitochondrial inheritance, although the mutant proteins retained half the GTPase activity of the wild-type protein. Although mitochondrial inheritance was not dependent upon Ca(2+) binding by the two EF-hands of Gem1p, a functional N-terminal EF-hand I motif was critical for stable expression of Gem1p in vivo. Our results suggest that basic features of Miro protein function are conserved from yeast to humans, despite differences in the cellular machinery mediating mitochondrial distribution in these organisms.

FIGURE 1.

Domain structure of yeast Miro GTPase, Gem1p. A, a schematic view of yeast Miro GTPase, Gem1p, showing the location of the two putative GTPase domains (GTPase I and II), two EF-hand motifs (EF-hand I and II), and the transmembrane segment (TM). The amino acid positions are indicated above the structure. B, sequences of predicted Gem1p GTP-binding sites (G1 motif) and EF-hand motifs aligned with Miro homologues. Shaded characters indicate residues mutated in this study. hMiro, human Miro; dMiro, Drosophila Miro. C, schematic representation of Gem1p on the mitochondrial outer membrane (20).

FIGURE 2.

GTP hydrolysis activity of Gem1p variants. A, GST-tagged WT (GST-Gem1p(1–616)) and mutant (S19N, S462N, and S19N/S462N) Gem1 proteins were incubated with α-³²P-labeled GTP at 30 °C for the indicated times, and the reactants were analyzed by TLC. Equimolar GST protein alone was used as a negative control. Position of α-³²P-labeled GTP and GDP are indicated by arrows. Right bottom panel, the samples used in the GTPase assay were resolved by 10% SDS-PAGE and stained with Coomassie Blue. B, the percentage of GTP hydrolysis over the indicated time course was calculated from the intensities of the α-³²P-labeled GTP and GDP signals. Filled circle, WT GST-Gem1p(1–616); open square, S19N mutant; open circle, S462N mutant; open triangle, S19N/S462N; cross, GST alone. C, substrate saturation experiments were carried out by incubating GST-Gem1p(1–616) variants (5 μm) at 30 °C with increasing concentrations of cold GTP (from 0 to 1000 μm) in the presence of 18 nm α-³²P-labeled GTP. The kinetic data are plotted versus the concentrations of GTP, and the symbols used are the same as in B.

FIGURE 3.

Ca²⁺ binding property of Gem1p variants. A, top, 10 μm purified WT GST-Gem1p(1–616) and its E225K/E354K mutant were incubated with 0.15 mm ⁴⁵Ca-labeled CaCl₂ and spotted onto PVDF membrane, and binding was detected by autoradiography. The binding specificity was confirmed by addition of 200 mm unlabeled CaCl₂ to the reaction. BSA was used as a negative control. Bottom, quantification of ⁴⁵Ca binding to WT and EF-hand mutant Gem1 proteins, and Coomassie Blue staining of the purified proteins used in this assay. B, similar to A except showing results for the single E225K and E354K mutant proteins. Error bars, S.D.

FIGURE 4.

Binding of guanine nucleotides to WT Gem1p(1–616). A, FRET from Trp residues to bound mant-GDP (left) or mant-GTPγS (right) in the WT Gem1p(1–616) complex. Fluorescence emission spectra of 1 μm WT Gem1p(1–616) in the presence (red) or absence (black) of either 5 μm mant-GDP or -GTPγS were recorded at 25 °C with 5 mm MgCl₂. Blue plots represent the spectrum of the energy transfer quenched by the addition of 10 mm EDTA and subsequently restored (green) by the addition of 20 mm MgCl₂ to the quenched solution, indicating that mant-nucleotides are bound to WT Gem1p(1–616). An excitation wavelength (λ_ex) of 295 nm was used for all spectra, and fluorescence intensities are shown in arbitrary units (AU). B, titration of mant-GDP in the presence (filled squares) or absence (open squares) of 1 μm WT Gem1p(1–616). The fluorescence intensity was monitored at λ_em = 445 nm with excitation at 295 nm. C, competition between mant-GDP and guanine nucleotides (GDP or GTP) for binding to WT Gem1p(1–616). Emission spectra of 1 μm WT Gem1p(1–616) with 5 μm mant-GDP alone plus increasing concentrations (1, 2, 4, 5, 10, 20, 40, and 100 μm) of either GDP (top) or GTP (bottom) were monitored at 25 °C. D, the percentage of mant-GDP bound to the WT Gem1p(1–616) was plotted versus increasing concentrations of GDP (open circles), GTP (filled squares), and ATP (filled triangles).

FIGURE 5.

Steady-state abundance of Gem1 WT and mutant proteins expressed in yeast. A, steady-state abundance of WT and mutant Gem1 proteins expressed from the native GEM1 promoter on a low copy (CEN) plasmid in a gem1Δ strain. B, steady-state abundance of WT and mutant Gem1 proteins expressed from the uninduced MET25 promoter on a low copy plasmid in a gem1Δ strain. Whole cell extracts separated by 8% SDS-PAGE were transferred to membrane and immunoblotted with affinity-purified anti-Gem1p polyclonal primary antibody. Protein bands were detected using a fluorescent IRDye 680-conjugated anti-rabbit secondary antibody followed by scanning on an Odyssey imaging system (Li-Cor Biosciences). The minus sign in the far left lane denotes empty vector. Extract loaded/lane in A is twice the amount loaded/lane in B.

FIGURE 6.

Mitochondrial inheritance function of WT and mutant Gem1 proteins. A and B, corresponding differential interference contrast and digital fluorescence images of a wild-type strain with normal mitochondrial inheritance. C–F, gem1Δmmr1Δ cells with reduced mitochondrial inheritance. G–J, gem1Δmmr1Δ cells with defective mitochondrial inheritance. Cells are labeled with a mitochondria-targeted form of GFP (mito-GFP) and exhibit aberrant mitochondrial morphology in gem1Δmmr1Δ (21). K, quantification of mitochondrial inheritance by medium and large buds in gem1Δmmr1Δ (black bars). The presence of any mito-GFP in the bud was scored as successful inheritance. n ≥ 100. Error bars, S.D. values from three independent experiments. Bar, 5 μm.