Structure-Function Analysis of the Yeast Mitochondrial Rho GTPase, Gem1p

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 Ca2+ 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.

In addition to serving as the powerhouses of eukaryotic cells, mitochondria play central roles in programmed cell death and apoptosis (1), aging (2), calcium homeostasis (3), and innate immune response to viral infection (4 -6). Mitochondria in many cell types are tubular and undergo cycles of homotypic fusion and fission, opposing processes that control organelle shape, copy number, and mitochondrial DNA maintenance (7,8). Optimal cell function also relies on pathways that control mitochondrial motility and distribution. Abnormalities in mitochondrial motility and distribution can cause severe defects in highly polarized cells, like motor neurons, where mitochondria delivered to synapses maintain local ATP and calcium levels (9,10).
Mitochondrial motility and distribution mechanisms are particularly critical during cell division because mitochondria in daughter cells cannot be generated de novo and instead arise by fission and inheritance of preexisting mitochondria from the mother cell. Movement of mitochondria is mediated by a set of conserved proteins in multicellular eukaryotes. In flies and mammals, mitochondria associate with mitochondria-specific kinesin motors (11)(12)(13) via an adaptor protein, called Milton (12,14), which binds in turn to a tail-anchored mitochondrial outer membrane receptor, mitochondrial Rho (Miro) 2 GTPase (15,16). Recently, Miro-independent targeting of human Milton to mitochondria has also been observed (17). In budding yeast, where most organelle movement occurs along actin filaments rather than microtubules, mitochondrial transport requires a type-V myosin motor (Myo2p) (18), two Myo2p-associating proteins (Mmr1p and Ypt11p) (18,19), and the single Miro ortholog, Gem1p (20,21). Although these molecular machineries differ with respect to the types of cytoskeletal tracks, motors, and accessory proteins employed, they converge at the point of the Miro/Gem1p receptor on the mitochondrial surface, underscoring the importance of this receptor in mitochondrial movement.
Members of the Miro family, including Gem1p, contain two GTPase domains (GTPase I and II) that flank two bipartite Ca 2ϩ -binding EF-hand motifs (EF-I and -II) (15,20) (Fig.  1, A and B). Because the C termini of these proteins are tailanchored in the outer mitochondrial membrane, all four domains are exposed to the cytoplasm (Fig. 1C). Genetic studies indicate that these domains are important for the function of Drosophila Miro (16), mammalian Miro (15,(22)(23)(24), and yeast Gem1p (20). However, the predicted activities of these domains have not been experimentally established, and it is not known whether the biochemical activities of the individ-ual domains are interdependent. In addition, it is unknown whether all four domains must be active in a single molecule for mitochondrial inheritance. In this study, we performed a structure/function analysis of the yeast Miro GTPase, Gem1p, and established that both GTPase domains are essential for mitochondrial inheritance. Conversely, Ca 2ϩ binding by the EF-hand motifs is not required for Gem1p function. Instead, a mutation that abolishes Ca 2ϩ binding by the N-terminal EF-I motif severely compromises protein stability.
Protein Expression and Purification-All proteins were expressed in E. coli strain BL21(DE3) cells. Overnight cultures (25 ml) were used to inoculate 1 liter of Luria broth (LB) medium and grown to log phase at 37°C. Overproduction of protein was induced by the addition of isopropyl 1-thio-␤-Dgalactopyranoside to a final concentration of 0.1 mM at 15°C for overnight induction. The next day, cells were collected by centrifugation (6,000 rpm for 15 min), and the pellets were stored frozen (Ϫ20°C) until purification. Bacterial pellets were resuspended in 50 mM Tris-buffered saline (pH 7.2) con-taining 300 mM NaCl, 5 mM MgCl 2 , 5 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF), lysed by sonication, and centrifuged at 15,000 rpm for 15 min to obtain a soluble fraction. After clarification, the GST-tagged proteins were affinity-purified on glutathione-Sepharose 4B columns (GE Healthcare) at 4°C. Proteins were eluted with 50 mM Tris-HCl buffer (pH 8.5) containing 300 mM NaCl, 5 mM MgCl 2 , and 20 mM reduced glutathione. After elution, all proteins were purified to Ͼ95% purity by gel filtration chromatography using a Sephacryl S-300 column (GE Healthcare) equilibrated with 50 mM Tris-HCl buffer (pH 7.2) containing 150 mM NaCl, 5 mM MgCl 2 , 5% (w/v) glycerol in the presence of 5 M GDP.
To remove the N-terminal GST tag from the Gem1p(1-616) construct, the fusion protein was dialyzed against 50 mM Tris-HCl buffer (pH 7.2) containing 150 mM NaCl, 5 mM MgCl 2 , 5 M GDP, and 1 mM DTT in the presence of GST-Precision protease (GE Healthcare) at 4°C (18 h) and then subjected to glutathione-Sepharose 4B chromatography to remove the protease and uncleaved protein. All protein concentrations were determined by absorbance at 280 nm in 6 M guanidine hydrochloride (28).
GTP Hydrolysis Assay-The GTP hydrolysis activity of GST-Gem1p variants (final concentration of 5 M) was assayed in 20 l of 50 mM Tris-HCl buffer (pH 7.2) containing 100 mM KCl, 5 mM MgCl 2 , 1% (w/v) glycerol, 10 M cold GTP, 5 M GDP, 1 mM DTT in the presence of 18 nM hot GTP (␣-32 P-labeled) at 30°C (without any free Ca 2ϩ ions in the reaction buffer). At each incubation time (0, 10, 20, 30, 40, 60, 90, and 120 min), the reaction was quenched by the addition of an equal volume of a stop solution (0.5% SDS, 10 mM EDTA, and 2 mM DTT) and heating at 65°C for 1 min. One microliter of each reaction was spotted onto a polyethyleneimine-cellulose thin layer chromatography (TLC) plate (Sigma-Aldrich) and resolved in 1 M formic acid and 0.5 M LiCl solution. The TLC plate was exposed to an imaging plate (Fuji Film, Tokyo, Japan), and the signal was detected by using an FLA 5100 phosphor imager (Fuji Film).
To determine the catalytic constant (K cat ) of GST-Gem1p variants, a 5 M concentration of each protein was incubated with various concentrations (0, 10, 20, 50, 100, 200, 500, 750, and 1000 M) of cold-GTP in the presence of 18 nM hot GTP. After incubation at 30°C, the reactions were spotted onto a TLC plate and analyzed as described above. The K cat from the GTP hydrolysis of GST-Gem1p variants was determined by Hanes-Woolf plot. In this assay, the GTP hydrolysis activity of WT Gem1p was also examined in the presence of 2 mM CaCl 2 to evaluate the effect of Ca 2ϩ binding in the enzymatic activity.
Nucleotide-binding Assay-To prepare the samples for nucleotide binding assays, the recombinant WT Gem1p(1-616) was dialyzed against 50 mM Tris-HCl buffer (pH 7.2) containing 5 mM EDTA, 150 mM NaCl, 5% (w/v) glycerol, and 1 mM DTT in order to generate nucleotide-free forms of the protein. After dialysis, 3 ml of the protein solution (1 M) was incubated with a 5 M concentration of either mant-GDP or -GTP␥S for 15 min at 25°C in the presence of 5 mM MgCl 2 . Binding of either mant-GDP or -GTP␥S to the WT Gem1p(1-616) was measured using a fluorescence spectrophotometer (JASCO FP-6300, Kyoto, Japan) with the excitation wavelength ( ex ) at 290 nm and the emission spectra ( em ) from 300 to 550 nm at 25°C. The emission spectra were collected with Spectra Manager software (JASCO Co.).
Determination of Equilibrium Dissociation Constants-Determination of equilibrium dissociation constants (K d ) of WT Gem1p(1-616) with either GDP or GTP by fluorescence measurement was performed as described previously (29) with minor modifications. Briefly, we determined the equilibrium dissociation constant of the WT Gem1p(1-616)⅐mant-GDP complex (K d mGDP ) prior to the determination of K d GDP and K d GTP . One micromolar WT Gem1p(1-616) solution was titrated with increasing amounts of mant-GDP (0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 M), and the apparent fluorescence intensity (Y app ) was calculated using the following equation, where F mGDP and F Gem1p where [Gem1p(1-616)] total is the total concentration of WT Gem1p(1-616). The K d mGDP and F Gem1p(1-616)⅐mGDP values were obtained by fitting the measured fluorescence data to the following equation, After estimating the K d mGDP , the equilibrium dissociation constants of GDP and GTP for WT Gem1p(1-616) were measured indirectly by competition with mant-GDP. The data fitting was performed using the program Mathematica 6 (Wolfram Research).
Protein Extraction from Yeast and Western Blotting-An alkaline extraction method (30) was used to prepare protein samples from whole yeast cells grown to log phase at 25°C in synthetic dextrose medium. Protein extracts prepared from strains expressing plasmid-borne Gem1p WT and mutant proteins were subjected to 8% SDS-PAGE (native GEM1 promoter, 25 l ϭ 1.0 A 600 cells/gel lane; MET25 promoter, 12.5 l ϭ 0.5 A 600 cells/lane). Separated proteins were transferred to nitrocellulose and incubated with affinity-purified anti-Gem1p polyclonal antibody (1:500 dilution) (Shaw laboratory). Following incubation with fluorescent secondary antibodies (IRDye 680 anti-rabbit; 1:5000 Li-Cor Biosciences, Lincoln, NE), proteins were detected and quantified using an Odyssey scanner and Odyssey 3.0 analysis software (Li-Cor Biosciences). A background band recognized by the anti-Gem1p antibody was used as an internal loading control in each lane (not shown).
Analysis of Mitochondrial Inheritance-Mitochondrial inheritance was scored at 25°C in strains expressing a matrixtargeted form of mito-GFP (pXY142-mtGFP plus) and grown to log phase (A 600 0.5-1.0) in dextrose-containing medium. Data reported are the average and S.D. from three or more independent experiments (n Ն 100).
Microscopy and Image Acquisition-Digital fluorescence and differential interference contrast microscopic images of cells were acquired using an Axioplan 2 deconvolution microscope (Carl Zeiss Microimaging, Inc.) as described previously (20). Images were processed using Zeiss Axiovision version 4.1 and assembled into figures using Adobe Photoshop CS and Adobe Illustrator CS using only linear adjustments of contrast and brightness.

Both Predicted Gem1p GTPase Domains Hydrolyze
Nucleotide-We bacterially expressed the cytosolic domain of wild-type GST-Gem1p (designated as GST-Gem1p(1-616); Fig. 1A), which lacks the C-terminal transmembrane domain, and assayed for its ability to hydrolyze GTP in vitro. As shown in Fig. 2, A and B, time-dependent GTP hydrolysis was observed in reactions containing GST-Gem1p(1-616) but not GST alone. Introduction of S19N or S462N substitution mutations into the GTPase I or II domains of GST-Gem1p(1-616), respectively, reduced their GTPase activities to a similar extent (Fig. 2B). GTP hydrolysis activity was completely abolished when the S19N or S462N substitutions were introduced into the same molecule (Fig. 2, A and B; S19N/S462N), establishing that both domains contribute to GTP hydrolysis by Gem1p.
We measured the initial rate of GTP hydrolysis to determine the K cat and K m of WT and mutant Gem1p variants. Kinetic analysis revealed that the K cat for the S19N/S462N variant was significantly slower than that of WT ( Fig. 2C and Table 1). By contrast, the individual S19N and S462N variants had more modest effects, reducing the catalytic rate of GTP hydrolysis relative to WT. Consistent with these findings, purified proteins containing only the GTPase I or II domains (Gem1p(1-200) and Gem1p(441-616)) exhibited K cat values of ϳ0.2 min Ϫ1 , only slightly less than that of WT (0.24 min Ϫ1 ) ( Table 1). Our biochemical analyses establish Gem1p as a member of the Ras GTPase superfamily, which is characterized by slower catalytic rates (31)(32)(33), rather than the Dynamin GTPase superfamily, which is characterized by faster catalytic rates (34 -36).
The EF-hand Motifs of Gem1p Bind to Calcium Ions-Calcium signaling mediates numerous cellular processes including mitochondrial motility (37). Although the EF-hand motifs of Miro GTPases are proposed to function as a calcium-sensitive switch (23,24,38), there is limited evidence that Miro GTPases physically bind calcium ions (39). To address this issue, we determined whether GST-Gem1p(1-616) containing WT or mutant (E225K or E354K) EF-hand motifs bound radioactive 45 Ca ions. In this assay, the WT protein, but not the negative control (BSA) and E225K/E354K proteins (right), tightly bound to 45 Ca ions (Fig. 3A). We also investigated the specificity of the protein⅐Ca 2ϩ complex and found that binding of radioactive calcium ions to WT Gem1p was abolished upon competition with an excess of unlabeled competitor, verifying the specificity of the interaction. By contrast, single E225K or E354K variants exhibited reduced binding (Fig. 3B), indicating that Gem1p is a calcium-binding protein and that its calcium binding activity resides in its two EF-hand motifs. Additional experiments revealed that the hydrolysis activity of WT Gem1p was not affected by the presence of free Ca 2ϩ ions in the reaction buffer; nor were changes observed in the GTPase activity of the E225K/E354K double mutant protein relative to that of WT protein ( Table 1), establishing that nucleotide binding and hydrolysis do not require Ca 2ϩ binding by the EF-hand motifs.
Nucleotide Binding to Gem1p-The two cytoplasmic GTPase domains in Gem1p are required for its function (20), and its GTP hydrolysis activity resides in these two domains (Fig. 2). To directly characterize their activity, we used a fluorescence assay with mant-substituted guanine nucleotides (mant-GDP and -GTP␥S) to measure the affinity of nucleotide binding. These assays employed WT Gem1 protein from which GST had been proteolytically removed (designated as WT Gem1p (1-616)). In the presence of Mg 2ϩ ions, WT Gem1p(1-616) bound to mant-GDP or mant-GTP␥S (Fig.  4A, red spectra), as indicated by substantial FRET from tryptophan residues in the protein ( em ϭ 332 nm) to the mantnucleotides ( em ϭ 438 nm) (black traces are negative controls showing FRET in the absence of protein). Chelating Mg 2ϩ ions in the reaction resulted in release of the bound mant-nucleotides from WT Gem1p(1-616) and loss of FRET (blue spectra). Most importantly, reintroducing excess free Mg 2ϩ ions resulted in decreased fluorescence intensity at 332 nm and a concomitant increase in intensity at 438 nm (green spectra), demonstrating that nucleotide binding was reversible and restored in a Mg 2ϩ -dependent manner. Titration experiments indicated that the equilibrium dissociation constant (K d ) for the mant-GDP was 0.27 M (Fig. 4B and Table 2).
Specificity of nucleotide binding in these experiments was established by showing that increasing concentrations of unlabeled GDP or GTP decreased the FRET signal generated by the WT Gem1p(1-616)⅐mant-GDP complex (Fig.  4C). Furthermore, competition for WT Gem1p(1-616) binding by added nucleotides was dependent on the guanine moiety; nucleotide exchange did not occur in the presence of ATP (Fig. 4D). Although GTP and GDP bound to WT Gem1p(1-616) with similar micromolar affinities, the protein had slightly higher affinity for GTP than for GDP ( Table 2).
The EF-hand I Motif of Gem1p Is Essential for Protein Stability-Mutation of Gem1p GTPase domains and EFhand motifs has the potential to destabilize the mutant proteins in vivo. Using single-copy plasmids, we expressed WT and mutant Gem1p proteins from the native GEM1 promoter in a gem1⌬ strain and compared steady-state protein levels by Western blotting. Although Gem1 proteins containing GTPase domain mutations (S19N, S462N, and S19N/S462N) were reproducibly detected in yeast whole cell extracts, their steady-state levels were slightly lower than WT protein (Fig. 5A). We did not detect proteins harboring the EF-I mutation E225K, although Gem1p abundance was unaf- fected by the EF-II mutation E354K (Fig. 5A). These data indicate that calcium binding by the N-terminal EF-hand I motif is important for Gem1p stability in vivo.
To increase protein expression, we cloned WT Gem1p and the mutant variants behind the MET25 promoter. When expressed from MET25 without induction, the steady-state abundance of WT, GTPase domain mutant, and E354K mutant proteins was ϳ80 -100-fold greater than that expressed from the native GEM1 promoter (Fig. 5B). 3 Most importantly, MET25 expression produced detectable steady-state levels of E225K-containing proteins (ϳ20-fold overexpressed), allowing functional analysis of these Gem1p variants in vivo (Fig. 5B). 3 Mitochondrial Inheritance Requires the GTPase Domains but Not Ca 2ϩ Binding to the EF-hand Motifs of Gem1p-We previously demonstrated that Gem1p and the myosin adaptor proteins Mmr1p and Ypt11p act in independent pathways to promote mitochondrial inheritance (21). Cells lacking any two of these proteins displayed mitochondrial inheritance defects such that mother cells produced daughter cells (buds) without mitochondria. This defective mitochondrial inheritance prevented release of the affected buds from mother cells and was correlated with growth defects in the double mutant strains. Strains lacking all three proteins/pathways were essentially inviable.
To determine the importance of Gem1p functional domains in mitochondrial inheritance, we scored the distribu-  Gem1p(1-616)) and mutant (S19N, S462N, and S19N/S462N) Gem1 proteins were incubated with ␣-32 P-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 ␣-32 P-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 ␣-32 P-labeled GTP and GDP signals.  tion of GFP-labeled mitochondria in a gem1⌬mmr1⌬ strain expressing WT and mutant Gem1p proteins from the uninduced MET25 promoter. In these studies, the detection of any amount of GFP-labeled mitochondria in buds was scored as successful inheritance. Cells in Fig. 6, B, D, and F, show examples of successful mitochondrial inheritance, whereas those in Fig. 6, H and J, show examples of defective mitochondrial inheritance. The gem1⌬mmr1⌬ double mutant displayed a strong inheritance defect in medium-and large-budded cells (only 56% of buds inherited mitochondria; Fig. 6K, vector).
(The residual 56% inheritance in this strain is provided by the intact YPT11 pathway.) Expression of WT Gem1p restored mitochondrial inheritance in this strain (95% inheritance; Fig.  6K). Expression of Gem1 proteins with mutations in one or both GTPase domains (S19N, S462N, and S19N/S462N) did not rescue the gem1⌬mmr1⌬ defect (54 -63% inheritance; Fig. 6K), consistent with the idea that GTP hydrolysis by both domains is required for Gem1p function. Significantly, mitochondrial inheritance was completely rescued by overexpressed Gem1 proteins with mutations in EF-I, EF-II, or both motifs (E225K, E354K, and E225K/E354K) (98 -99% inheritance; Fig. 6K). When combined with our calcium binding studies (Fig. 3), these results provide evidence that Ca 2ϩ binding by the two Gem1p EF-hand motifs is not essential for mitochondrial inheritance. In control experiments, scoring mitochondrial inheritance after a 4-h induction of the MET25 promoter did not alter the ability of the WT or mutant proteins to rescue gem1⌬mmr1⌬ inheritance defects (data not shown). Moreover, expression and overexpression of WT or mutant Gem1 proteins in a wild-type strain did not cause dominant inheritance defects or an excessive inheritance phenotype (accumulation of excess mitochondria in buds and/or depletion of mitochondria from the mother cell).

DISCUSSION
Miro proteins are recognized as key components of the mitochondrial distribution machinery in yeast, plants, invertebrates, and mammals (7, 15, 16, 20 -24, 38 -40). Although genetic analyses suggest that both GTPase domains and EFhands are required for Miro function, direct evidence for the activities of these domains is limited. Here we show that the predicted GTPase domains and EF-hand motifs in the yeast Miro, Gem1p, hydrolyze GTP and bind Ca 2ϩ ions, respectively. By monitoring the ability of mutant proteins to rescue mitochondrial inheritance defects in vivo (Fig. 6), the nucleotide hydrolysis activities of both GTPase domains were shown to be essential for Gem1p function. Mutations that blocked Ca 2ϩ binding to one or both EF-hand motifs did not impair mitochondrial morphology or block mitochondrial inheritance. Thus, if Ca 2ϩ binding exerts a regulatory impact, it most likely negatively regulates the function of Gem1p in mitochondrial inheritance. We also observed that mutation of the N-terminal EF-I motif (E225K) had a dramatic effect on Gem1 protein stability (Fig. 5). Mitochondrial inheritance was unaffected in cells overexpressing WT, GTPase mutant, or EF-hand mutant Gem1 proteins, indicating that Gem1p activity is not rate-limiting in vivo and that mutant proteins do not dominantly interfere with the function of WT Gem1p or its cellular binding partners. These results address outstanding issues regarding the classification of the Gem1p GTPase domains, the roles of Ca 2ϩ binding to Gem1p in vivo, and the conserved functions of Miro proteins.
Miro proteins were originally classified as Rho GTPases (15). A later study noted that the Gem1p N-terminal GTPase domain lacked a Rho-specific sequence insert, and the C-terminal GTPase domain sequence was not closely related to the Ras or Rho GTPase families (20). More recently, Miro pro- teins have been reclassified as a subfamily of the Ras GTPase superfamily (31,38,41). Consistent with this new classification, we showed that the rates of GTP hydrolysis by truncated Gem1 proteins containing only GTPase I or GTPase II domains are slow, with K cat of ϳ0.2 min Ϫ1 (Table 1). This poor intrinsic GTPase activity suggests that both Gem1p GTPase domains, like other members of the Ras family, utilize accessory factors in vivo to increase the rate of hydrolysis. We also observed that Gem1 proteins containing single GTPase domain mutations retained GTP hydrolysis activity (Fig. 2), indi-cating that the activities of the GTPase I and II domains are not interdependent. Despite this residual activity, single GTPase I and GTPase II mutations eliminated the ability of Gem1p to rescue mitochondrial inheritance defects in yeast. Additional experiments demonstrate that Gem1p with a GTPase I domain mutation could not be complemented in trans by Gem1p containing a GTPase II domain mutation. 4 These combined results demonstrate that normal Gem1p function requires two active GTPase domains in a single polypeptide chain. Finally, no significant difference in GTP hydrolysis was detected when free Ca 2ϩ ions were included in the reaction buffer or when Gem1p contained mutations in both EF-hand motifs ( Table 1), suggesting that Ca 2ϩ binding does not stimulate or inhibit the GTPase activity of one or both GTPase domains in this assay.
The EF-hand motifs of Miro proteins have never been shown to bind calcium independently; nor has the effect of Ca 2ϩ binding on protein stability been evaluated. Here we demonstrate that point mutations in EF-I or EF-II of Gem1p reduced Ca 2ϩ binding to a similar extent. In addition, mutation of both EF-hand motifs in a single Gem1 polypeptide chain completely abolished Ca 2ϩ binding (Fig. 3A). Because the 45 Ca binding assay does not detect low affinity Ca 2ϩ binding (42), both EF-hands in Gem1p probably bind calcium ions with high affinity. Importantly, mutation of the EF-I motif significantly reduced the steady state abundance of Gem1p in vivo (Fig. 5). Although cellular conditions that reduce or abolish Ca 2ϩ binding to EF-I in Gem1p have not been identified, such conditions are predicted to cause loss of protein function, presumably due to changes in protein stability and/or turnover. Consistent with this prediction, the K cat for Gem1p GTPase activity is not altered by mutation of the EF-hands (Table 1) although the K m value is slightly increased. Gem1 proteins containing EF-hand mutations may require a higher GTP concentration to achieve a given reaction velocity because they have a less ordered structure.
Using overexpression constructs, we stably produced EFhand mutant proteins and demonstrated that Ca 2ϩ binding by Gem1p is not necessary for its function in mitochondrial inheritance. This result is consistent with current models for Miro protein regulation, in which Ca 2ϩ binding to EF-hand motifs negatively regulates Miro interaction with Milton adaptor/kinesin motor complexes that promote mitochondrial movement on microtubules (24,39). In contrast to studies performed in other systems, overexpression of WT or GTPase/EF-hand mutant forms of Gem1p had no discernable effect on mitochondrial distribution or morphology in yeast (20) (this study). Considering that yeast lack a Milton homolog and that yeast mitochondria move on actin filaments rather than microtubules, it seems likely that Miro protein function is regulated differently in distinct organisms and/or cell types. Identification of Gem1p binding partners in yeast will provide a means to study additional modes of Gem1p regulation and function.