The Primary Defect Associated with Loss of MGM1 Function Is Fragmentation of Mitochondrial Reticuli
Previous work showed that MGM1
is required for wild-type mitochondrial morphology and respiratory competence. Specifically, mtDNA loss and mitochondrial aggregation and inheritance defects were observed in temperature sensitive mgm1
cells (Guan et al. 1993
; Jones and Fangman 1992
; Shepard and Yaffe 1999
). To gain more insight into the exact role of MGM1
in morphology maintenance, we analyzed new temperature-sensitive alleles of MGM1
isolated in a screen for conditional mutants that are unable to maintain mtDNA (Meeusen et al. 1999
To determine the primary defect associated with loss of MGM1
function, we focused on one conditional allele of MGM1
. This allele was chosen because analysis of our collection of mgm1
mutants indicated that mgm1-5
cells exhibited the greatest and most rapid phenotypic change upon shifting to nonpermissive temperature. Specifically, under permissive conditions, no difference in the generation of respiratory incompetent colonies was observed between mgm1-5
and wild-type cells, indicating that Mgm1p is functional under these conditions (99% respiratory competent, n
= 100). In contrast, under restrictive conditions, 100% of mgm1-5
cells were respiratory incompetent and devoid of mtDNA as compared with 3% of wild-type cells, indicating a complete loss of MGM1
= 100). In heterozygous mgm1-5 MGM1
phenotypes were recessive, indicating that the mutation causes a loss of MGM1
function at nonpermissive temperature. The MGM1
locus in mgm1-5
cells contains a single point mutation that changes G408 to D408. This residue is contained within the GTPase domain of the protein, consistent with previously published observations indicating its importance for MGM1
function (Shepard and Yaffe 1999
To gain more insight into MGM1 function, we examined mitochondrial morphology in mgm1-5 cells using mito-GFP. Under permissive conditions, the majority of mgm1-5 cells contained reticular mitochondria, similar to that observed in wild-type cells (, compare B to D). Within 20 min after shifting to the nonpermissive temperature, mitochondrial reticuli fragmented into many smaller mitochondria that remained distributed at the cell cortex in mgm1-5 cells (, compare D to F). Extended exposure (60 min) of mgm1-5 cells to nonpermissive temperature resulted in aggregation of mitochondrial fragments ( H). This terminal morphological phenotype resembles the mitochondrial morphology defect observed in Δmgm1 cells (, compare H to J). Thus, the primary phenotype associated with loss of MGM1 function is mitochondrial fragmentation and a secondary phenotype is aggregation of these mitochondrial fragments (see K for quantification).
Figure 1 The primary phenotype resulting from loss of MGM1 function is fragmentation of mitochondrial reticuli. Mito-GFP was used to visualize mitochondria in wild-type (A and B), mgm1-5 (C and D), and Δmgm1 (I and J) cells grown overnight at 25°C (more ...)
A previous study reported a mitochondrial inheritance defect in mgm1
cells and suggested that Mgm1p might be involved in the movement of mitochondria into daughter cells (Shepard and Yaffe 1999
). We observed mitochondrial inheritance defects in mgm1-5
cells only after prolonged exposure to nonpermissive temperature. Specifically, 5% (n
= 130) of buds lacked mitochondria after a 20-min incubation at 37°C versus 45% (n
= 73) at 60 min. However, the majority of cells that lacked mitochondria in the bud contained a single mitochondrial aggregate. In addition, in a small percentage of cells, the mitochondrial aggregate was present in the bud and absent from the mother, indicating that in these cases the aggregate was transported into the yeast bud. Thus, Mgm1p does not function directly to mediate mitochondrial movement into daughter cells.
Mitochondrial Fusion Is Blocked in mgm1-5 Cells during Mating
Both mitochondrial fragmentation and mtDNA loss phenotypes observed in mgm1-5
cells are similar to the phenotypes associated with loss of FZO1
function (Hermann et al. 1998
). In fzo1-1
cells, mitochondrial fragmentation results from a block in fusion and unopposed, ongoing mitochondrial fission. To determine if MGM1
, like FZO1
, has a role in mitochondrial fusion, we examined mitochondrial fusion in mgm1-5
cells during mating. Mitochondrial fusion was assayed as previously described by labeling mitochondria in haploid cells of opposite mating type with either a mito-GFP or a covalent vital probe, MitoTracker (Nunnari et al. 1997
). Mitochondrial fusion was assessed by examining the distribution of these probes in large-budded zygotes formed at both permissive and nonpermissive temperature. Consistent with previously published observations, in wild-type zygotes, both haploid-derived mitochondrial probes were colocalized at permissive and nonpermissive temperatures indicating that haploid mitochondria had fused and their contents had mixed (Nunnari et al. 1997
; , A–D; ). Haploid mitochondrial content mixing also occurred in the majority of the mgm1-5
zygotes formed at the permissive temperature (, E–H; ). In contrast, at the nonpermissive temperature, mitochondrial reticuli fragmented in mgm1-5
zygotes and the resulting fragments failed to fuse, even though mitochondria were closely associated with one another (, I–L; ). This fusion defect is similar to that observed in fzo1-1
zygotes, supporting the idea that MGM1
function is important for mitochondrial fusion (Hermann et al. 1998
; , M–P; ).
Figure 2 Mitochondrial fusion is blocked in mgm1-5 cells during mating. Cells of opposite mating type were grown to log phase at 25°C, labeled with either mito-GFP or MitoTracker and mated at 25°C (E–H) or 37°C (A–D, I–P). (more ...)
Quantification of Fusion in Large-budded Zygotes during Mating
Deletion of DNM1 in mgm1-5 Cells Blocks Mitochondrial Fragmentation and Restores Mitochondrial Fusion during Mating
To further test the hypothesis that fragmentation of mitochondrial reticuli in mgm1-5 cells is the result of a block in fusion, we determined whether mitochondrial tubules were restored in mgm1-5 cells under conditions where mitochondrial fission is abolished. To block mitochondrial fission in mgm1-5 cells, we deleted DNM1, which encodes a dynamin-related GTPase required for mitochondrial division. Mitochondrial morphology was examined in mgm1-5 Δdnm1 cells at permissive and nonpermissive conditions using mito-GFP. Under permissive conditions, where Mgm1p is functional, mitochondrial net-like structures are observed in mgm1-5 Δdnm1 cells (). These net-like mitochondrial structures are characteristic of Δdnm1 cells and likely arise because mitochondrial tubules fuse and new tubule ends cannot be generated by fission ( and ; ). At the nonpermissive temperature in mgm1-5 Δdnm1 cells, where mitochondrial fragmentation is observed in mgm1-5 cells, mitochondrial tubules and net structures are maintained (, compare C and D to G and H; ). Thus, abolishing Dnm1p-dependent mitochondrial fission blocks mitochondrial fragmentation resulting from loss of MGM1 function. In addition, the mgm1-5 glycerol growth and mtDNA loss phenotypes observed at nonpermissive temperature are suppressed in mgm1-5 Δdnm1 cells (not shown). Deletion of DNM1 in fzo1 cells also has been shown to block mitochondrial fragmentation and mtDNA loss.
Quantification of Mitochondrial Morphology at 37°C
Figure 3 Deletion of DNM1 blocks mitochondrial fragmentation in mgm1-5 cells. Cells were grown to log phase at 25°C and shifted to 37°C for 40 min. Mitochondria were visualized using mito-GFP by fluorescence confocal microscopy in wild-type (A (more ...)
Although these data suggest that MGM1
might play a role in the fusion process, the structural similarity of Mgm1p to dynamin-like proteins suggests that it is involved in membrane remodeling and/or fission events. Thus, we considered the alternative explanation, that mitochondrial fragmentation in mgm1-
5 cells results from an increase in Dnm1p-dependent mitochondrial fission. In this scenario, mitochondrial fragments might fail to fuse because of their geometry/structure and not as a primary consequence of loss of MGM1
function. To test this possibility, we assayed mitochondrial fusion during mating in mgm1-5
cells, where deletion of DNM1
blocks mitochondrial fragmentation and restores mitochondrial tubular structures ( and ; ). As shown previously, deletion of DNM1
has no effect on mitochondrial fusion during mating, consistent with its role in fission (Bleazard et al. 1999
; , A–D; ). Thus, as expected at the permissive temperature where MGM1
is functional, mitochondrial fusion and content mixing occurred in zygotes formed from mgm1-5
haploids (). In contrast to mgm1-5
cells at nonpermissive temperature, where both mitochondrial fragmentation and a block in mitochondrial fusion are observed, deletion of DNM1
cells restored mitochondrial fusion during mating (, E–H; ). Thus, the presence of mitochondrial tubular and net structures in mgm1-5
cells correlates with the restoration of mitochondrial fusion (). In contrast, FZO1
function is still required for mitochondrial fusion, even when tubules are restored in fzo1-1
zygotes, consistent with Fzo1p's proposed direct role in the fusion process (Bleazard et al. 1999
; , I–L; ).
Figure 4 Mitochondrial fusion does not require MGM1 function. Mitochondrial fusion was assessed as described in . Mitochondria in homozygous zygotes formed at 37°C from Δdnm1 (A–D), mgm1-5 Δdnm1 (E–H), fzo1-1 Δ (more ...)
To test whether restoration of mitochondrial fusion in mgm1-5
zygotes was FZO1
-dependent and not due to activation of an alternate fusion pathway, we examined mitochondrial fusion in the mgm1-5 fzo1-1
triple mutant. Morphologically, mitochondria in these triple mutant cells were similar to those in both Δdnm1
cells (, M–P). As expected, at permissive temperature, mitochondria fused in zygotes formed from mgm1-5 fzo1-1
haploids (). However, at nonpermissive temperature, mitochondria failed to fuse in mgm1-5 fzo1-1
zygotes, confirming previous evidence indicating a direct role of Fzo1p in mitochondrial fusion (Hermann et al. 1998
; , M–P; ). Taken together these observations indicate that MGM1
does not play a direct role in mitochondrial fusion.
Mgm1p Is Localized to the Mitochondrial Intermembrane Space
Given that MGM1
does not play a direct role in mitochondrial fusion, we suggest three possible explanations for the mitochondrial fragmentation observed in mgm1-5
cells. In wild-type cells, Mgm1p may function indirectly in mitochondrial fusion by remodeling and/or maintaining mitochondrial membranes in fusion competent tip and tubular structures. Alternatively, given that both Mgm1p and Dnm1p are dynamin-related GTPases predicted to assemble into oligomeric structures, Mgm1p may interact directly with Dnm1p to negatively regulate mitochondrial fission. This latter possibility is potentially supported by a recent report that Mgm1p is localized to the mitochondrial outer membrane (Shepard and Yaffe 1999
). However, experiments examining the S
Mgm1p homologue, Msp1, suggested that this protein is localized inside mitochondria, specifically in the matrix compartment (Pelloquin et al. 1999
). A third possibility is that Mgm1p may function in inner membrane organization and/or fission and that these events may be coordinated with outer membrane fission. In this scenario, loss of MGM1
function might stimulate Dnm1p-dependent outer membrane fission, resulting in mitochondrial fragmentation. To help distinguish between these possibilities, we reexamined the submitochondrial localization of Mgm1p using subcellular fractionation, indirect immunofluorescence, and cryoimmunoelectron microscopy analyses.
To detect and examine its subcellular localization, we epitope-tagged Mgm1p. Neither NH2- or COOH-terminal tagged Mgm1p complemented the mitochondrial morphology and growth defects of mgm1 cells (not shown). A tagged version of Mgm1p that retained function was created by inserting three tandem copies of the HA epitope (3XHA) within the coding sequence between amino acids 216–217, directly proceeding the predicted GTPase domain (Mgm1:3XHAp). In a strain where Mgm1:3XHAp replaced the wild-type MGM1 locus (JSY2519), mitochondrial morphology was indistinguishable from wild-type cells (n = 443).
Western blot analysis of extracts made from JSY2519 with anti-HA antibodies detected a predominant 92-kD species ( A, lane 1). In contrast, no species were detected by Western blotting in the absence of tagged Mgm1p, indicating that anti-HA antibodies specifically recognize Mgm1:3XHAp ( A, lane 2).
Figure 5 Mgm1p is a mitochondrial intermembrane space protein peripherally associated with the inner membrane. Whole cell extracts of JSY836 and JSY2519 were prepared as described in Materials and Methods and analyzed by SDS-PAGE and Western blotting with indicated (more ...)
To examine Mgm1p's subcellular localization, JSY2519 extracts were fractionated by differential centrifugation and analyzed by SDS-PAGE and Western blotting. Consistent with the previously reported mitochondrial localization of Mgm1p, Mgm1:3XHAp was highly enriched in mitochondrial pellet ( B, lane 2). In addition to the predominant 92-kD form of Mgm1:3XHAp, we also detected a less abundant, slower migrating 116-kD form of Mgm1:3XHAp in enriched mitochondrial fractions ( B, lane 2). These data are in agreement with a previous study of Mgm1p localization, where two forms of Mgm1p were detected with antibodies raised against a COOH-terminal Mgm1p-derived peptide (Shepard and Yaffe 1999
). Both the 92- and 116-kD forms of Mgm1:3XHAp are localized to the same mitochondrial compartment (see below).
Indirect immunofluorescence of JSY2519 cells using anti-HA antibodies revealed that Mgm1p:3XHAp was localized to reticular structures at the cell cortex ( B). Mgm1:3XHAp staining colocalized with structures labeled with the covalent vital mitochondrial fluorescent probe, MitoTracker ( and ). Thus, localization of Mgm1p by both biochemical and indirect immunofluorescence indicate that it is a mitochondrial protein, consistent with previous observations (Shepard and Yaffe 1999
Figure 6 Mgm1:3xHAp localizes to mitochondria in vivo. JSY2519 cells were grown in YPD to log phase, labeled with MitoTracker (B), processed for indirect immunofluorescence with anti-HA (C), and were imaged using fluorescence confocal microscopy as described. (more ...)
We examined the submitochondrial localization and topology of Mgm1:3XHAp by treating isolated mitochondria with exogenous proteases. In intact mitochondria, both the 92- and 116-kD forms of Mgm1:3XHAp were inaccessible to even high concentrations of proteinase K (PK; B, lanes 2–4) and trypsin (not shown). Under these conditions, however, the outer mitochondrial marker, Fzo1p, was digested completely, indicating that the outer membrane was fully accessible to protease ( B, lanes 2–4). In addition, both intermembrane space (AAC and Tim23p) and matrix (KDH) marker proteins were protected from proteolysis, confirming that the mitochondrial outer and inner membranes were intact ( B, lanes 2–4). When both the inner and outer mitochondrial membranes were disrupted by treatment with detergent or by sonication, complete proteolysis of Mgm1:3XHAp and marker proteins in all mitochondrial compartments was observed ( B, lane 5, and C, lanes 1 and 2). These results indicate that Mgm1p is not a component of the mitochondrial outer membrane and is localized inside mitochondria. Thus, Mgm1p does not directly interact with Dnm1p to regulate mitochondrial outer membrane fission.
To determine whether Mgm1p resides in the intermembrane space or matrix compartment, we converted intact mitochondria to mitoplasts by selectively rupturing the mitochondrial outer membrane by hypoosmotic shock. Upon osmotic shock, a significant fraction of the soluble intermembrane space protein, cytochrome b2, was released into the supernate fraction after centrifugation of treated mitochondria ( D, lanes 1 and 2). In contrast, the inner membrane markers, AAC and Tim23p, and the soluble matrix protein, KDH, was recovered in the pellet fraction, indicating that the inner membrane of the mitoplasts remained intact ( D, lanes 1 and 2). Both forms of Mgm1:3XHAp also were recovered in the pellet fraction, indicating that they are either membrane-associated or soluble in the matrix.
To determine if Mgm1:3XHAp is localized to the matrix compartment or membrane-associated and exposed to the intermembrane space, we treated mitoplasts with PK. In mitoplasts, the matrix marker, KDH, was protected from PK digestion, indicating that the inner membrane was impermeant to protease and intact ( D, lanes 2–4). Although both AAC and Tim23p are inner membrane proteins, they contain epitopes exposed to the intermembrane space. Thus, as predicted, both were accessible to PK in mitoplasts ( D, lanes 2–4). Under these conditions, both forms of Mgm1:3XHAp behaved exactly like these intermembrane space markers and were also sensitive to PK digestion in mitoplasts ( D, lanes 2–4). In addition, identical results were observed for both forms of Mgm1:3XHAp and marker proteins when mitoplasts were treated with trypsin (not shown), indicating that the substrate specificity did not influence the susceptibility to proteolysis. These results indicate that both forms of Mgm1p are not localized to the matrix compartment, but instead, are membrane-associated and exposed to the intermembrane space.
To determine whether Mgm1p is associated with the outer or inner mitochondrial membrane, we examined Mgm1:3XHAp's localization by immunoelectron microscopy using anti-HA antibodies. Immunogold-labeling was performed as described on cryosections of JSY2519 cells (Bleazard et al. 1999
). Consistent with data from both indirect immunofluorescence and biochemical analyses, the majority of gold particles were found associated with mitochondrial structures (90%, n
= 71; , see m). Further analysis of mitochondrial-associated gold particles indicates that the vast majority were found within the interior of the organelle and, in most cases, clearly associated with the inner membrane (95%, n
= 64; , see arrows). This inner membrane association is especially apparent in sections where gold particles are associated with inner membrane folds or cistae (, see cr and arrow). In addition, we did not observe gold particles dispersed throughout mitochondria, unlike the distribution reported for Msp1p, the S
Mgm1p homologue (Pelloquin et al. 1999
Figure 7 Mgm1p is associated with the inner membrane. JSY2519 cells were grown in YPD to log phase and processed for cryoimmunoelectron microscopy using anti-HA. Mitochondrial profiles (m) from JSY2519 cells are shown (A–F). Arrows indicate gold particle (more ...)
To determine whether Mgm1p is an integral inner membrane protein, we examined Mgm1p's sensitivity to sodium carbonate extraction. Mitochondria containing Mgm1:3XHAp were extracted with 0.1 M Na2CO3 (pH 10.5) or mock-treated and fractionated into supernate and pellet fractions by centrifugation ( E, lanes 1–4). As expected, the inner membrane protein Tim23p was resistant to sodium carbonate extraction and was quantitatively recovered in the pellet fraction ( E, compare lanes 3 and 4). In contrast, both forms of Mgm1:3XHAp and the soluble intermembrane space protein cytochrome b2 were released into the supernatant upon sodium carbonate treatment, but not in control samples treated with buffer ( E, lanes 1–4). Based on this analysis, Mgm1:3XHAp is peripherally associated with the inner membrane, and not an integral membrane protein.