mmm1p spans both the outer and inner mitochondrial membranes

Kondo-Okamoto et al.

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Mmm1p Spans Both the Outer and Inner Mitochondrial Membranes, and

Contains Distinct Domains for Targeting and Foci Formation

Noriko Kondo-Okamoto, Janet M. Shaw*, and Koji Okamoto*

Department of Biology, University of Utah, Salt Lake City, UT 84112

*To whom correspondence should be addressed:

Department of Biology, University of Utah

257 South 1400 East, Salt Lake City, UT 84112

Tel.: (801) 585-6205 (to J.M.S.) / (801) 587-9209 (to K.O.)

Fax: (801) 581-2174

E-mail: [email protected] (to J.M.S.) / [email protected] (to K.O.)

Running Title: Mmm1p Is a Double Membrane-spanning Protein

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 12, 2003 as Manuscript M308436200 by guest on M

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SUMMARY

In the yeast Saccharomyces cerevisiae, the integral membrane protein Mmm1p is required for

maintenance of mitochondrial morphology and retention of mitochondrial DNA (mtDNA).

Mmm1p localizes to discrete foci on mitochondria that are adjacent to mtDNA nucleoids in the

matrix, raising the possibility that this protein plays a direct role in organizing, replicating or

segregating mtDNA. Although Mmm1p has been shown to cross the outer membrane with its C-

terminus facing the cytoplasm, the location of the N-terminus has not been resolved. Here we

show that Mmm1p spans both the outer and inner mitochondrial membranes, exposing its N-

terminus to the matrix. Surprisingly, deletion of the N-terminal extension decreased steady-state

levels of the Mmm1 protein, but did not affect mitochondrial morphology or mtDNA

maintenance. Moreover, expression of Neurospora crassa MMM1, which naturally lacks a long

N-terminal extension, substituted for loss of Mmm1p in budding yeast. These results indicate

that the matrix-exposed portion of Mmm1p is not essential for mtDNA nucleoid maintenance.

Additional studies revealed that the transmembrane segment and C-terminal domain of Mmm1p

are required for foci formation and mitochondrial targeting, respectively. Our data suggest that

Mmm1p’s double membrane-spanning topology at the membrane contact site is critical for

formation of tubular mitochondria.

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INTRODUCTION

Mitochondria are dynamic organelles that change size, shape and distribution in response to

changing cellular needs (1-4). Because mitochondria are essential and cannot be synthesized de

novo, most cell types employ cytoskeletal elements and molecular motors to partition the

organelles into newly formed daughter cells during division (5). Studies performed in a variety of

organisms including budding yeast reveal that maintenance of proper organelle morphology is

critical for mitochondrial function. In Saccharomyces cerevisiae, mitochondria form highly

branched tubular networks that continually divide and fuse (6-10). Mutations in a variety of

nuclear genes disrupt wildtype mitochondrial morphology, converting the tubular network into

small fragments, aggregates, large spheres, collapsed tubules, nets, or other aberrant shapes (11).

In some cases, these morphological changes lead to loss of mitochondrial DNA (mtDNA) and

defective respiratory activity (12-14). For example, mitochondria fragment and lose mtDNA in

cells lacking the large GTPase Fzo1p (15,16) or Ugo1p, (17) two integral outer membrane (OM)

proteins that mediate mitochondrial fusion. Similar mtDNA loss is observed in cells lacking the

intermembrane space (IMS) GTPase Mgm1p (18-21) required for mitochondrial fusion and inner

membrane (IM) remodeling, and the IM Rhomboid-type protease Mdm37p/Pcp1p/Rbd1p (11,22-

24) required for the biogenesis of Mgm1p. In the examples cited above, mtDNA loss is thought

to be a secondary consequence of changes in mitochondrial shape, since there is currently no

evidence that these proteins interact directly or indirectly with mtDNA nucleoids (mtDNA-

protein complexes). A second class of mutations that converts tubular mitochondria into large

spheres also leads to mtDNA loss. These mutations affect three integral OM proteins, called

Mdm10p (25), Mdm12p (26), and Mmm1p (27), which are thought to form a complex. Roles for

this complex in cytoskeletal attachment and mtDNA nucleoid maintenance have been proposed.


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The function of the Mmm1 protein has been studied in some detail. S. cerevisiae Mmm1p is

predicted to have a single transmembrane (TM) segment of ~25 residues (amino acids 92-116)

(27). Cells lacking functional Mmm1p display defects in mitochondrial motility and inheritance,

and mitochondria isolated from these cells are defective in ATP-sensitive actin-binding activity

in vitro (28,29). Based on these observations, it was proposed that Mmm1p is required for

interactions of mitochondria with the actin cytoskeleton. When tagged at the C-terminus with the

green fluorescent protein (GFP), Mmm1p-GFP localizes as discrete foci on the surface of

mitochondria (30). Strikingly, these Mmm1p-GFP foci appear adjacent to mtDNA nucleoids in

the matrix (30). Additional biochemical data suggest that Mmm1p resides at contact sites

between the inner and outer mitochondrial membranes (30). These findings raise the possibility

that Mmm1p is connected either directly or indirectly to mtDNA nucleoids.

Studies of the filamentous fungus Neurospora crassa MMM1 (NcMMM1) suggest that the

cellular role of this protein varies in different cell types. In N. crassa, mitochondria form tubular

arrays that co-align with microtubules rather than actin filaments (31). Unlike S. cerevisiae, N.

crassa cannot survive in the absence of mitochondrial respiration, and loss of mtDNA results in

lethality. Although NcMMM1 is also required for maintenance of normal mitochondrial

morphology, it is dispensable for interactions with microtubules (32,33). Moreover, the Mmm1

protein is not essential for mtDNA stability in N. crassa, since loss-of-function mutations are not

lethal (32). Thus, while the role of Mmm1p in mitochondrial morphology maintenance has been

conserved during evolution, its roles in cytoskeletal association and/or mtDNA retention may

have been altered or lost.

The phenotypes observed in mmm1 mutants suggest that Mmm1p contains distinct domains

with different functions. A better understanding of Mmm1p structure, topology and domain


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function may reveal how this protein carries out activities within the cell. S. cerevisiae Mmm1p

contains one predicted TM segment that spans the outer mitochondrial membrane leaving the C-

terminal domain in the cytoplasm (27). Although the N-terminus faces the interior of the

organelle, its exact location (IMS or matrix) has not been determined. In addition, the specific

functions of the Mmm1p N-terminal extension, TM segment and C-terminal domain have not

been explored. In this study, we determined the submitochondrial location of the Mmm1p N-

terminus, and identified domains important for mitochondrial targeting and foci formation. In

vivo site-specific cleavage and in vitro protease protection assays revealed that the N-terminus of

Mmm1p is exposed to the matrix, a topology that requires the protein to span both the outer and

inner mitochondrial membranes. Although deletion of the N-terminal extension resulted in

reduction of steady-state Mmm1p protein levels, it did not affect protein targeting to the

organelle, maintenance of mitochondrial morphology, or mtDNA retention. Moreover, the

mtDNA loss phenotype of the S. cerevisiae mmm1 null (mmm1 ) mutant could be rescued by

expressing NcMMM1, which naturally lacks a long N-terminal extension. These findings

indicate that the matrix-exposed, N-terminal extension of Mmm1p does not play a direct role in

mtDNA maintenance. We further show that the TM segment and C-terminal domain play

essential roles in Mmm1p foci formation and mitochondrial targeting, respectively. These

combined results suggest that Mmm1p’s double membrane-spanning topology at the membrane

contact site is crucial for establishment and maintenance of tubular mitochondrial morphology.

Implications of these findings with respect to mtDNA maintenance and cytoskeletal attachment

are discussed. Models for the double-membrane spanning topology of Mmm1p are also

presented.


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EXPERIMENTAL PROCEDURES

Yeast Strains and Growth Conditions—All strains are derivatives of the FY10 strain (34).

Standard genetic methods were used to grow, transform and manipulate yeast (35) and bacterial

(36) strains. Strains used in this study include: JSY1825 (MAT , his3 200, leu2 1, lys2 -202,

trp1 63, ura3-52) and NOY84 (MAT , his3 200, leu2 1, lys2 -202, trp1 63, ura3-52,

mmm1 ::HIS3, [pRS416-MMM1-His]). Plasmids containing MMM1 constructs were introduced

by plasmid shuffle into NOY84. Yeast transformants were grown at 30°C in SC media or on

dropout plates containing one of the following carbon sources: 2% dextrose (SD), 2% glycerol

(SGly), 2% raffinose (SR), or 2% galactose plus 2% raffinose (SGalR).

Plasmid Construction—For pRS416-MMM1-His and pRS315-MMM1-His, a BamHI-EcoRV

fragment encoding the 0.52 kb MMM1 5’-UTR and Mmm1p (amino acids 1-426) plus 8xHis,

and an EcoRV-XhoI fragment encoding the 0.45 kb MMM1 3’-UTR were generated by PCR and

cloned into the BamHI-XhoI sites of the low-copy yeast vectors pRS416 and pRS315,

respectively (American Type Culture Collection). For pRS315-mmm1 (2-75)-His, pRS315-

mmm1 (2-90)-His, and pRS315-mmm1 (2-120)-His, the 1.1 kb BamHI-SalI fragment of

pRS315-MMM1-His was replaced by a PCR-generated BamHI-SpeI fragment encoding the 0.52

kb MMM1 5’-UTR plus ATG and a PCR-generated SpeI-SalI fragment encoding Mmm1p

(amino acids 76-190), (amino acids 91-190), and (amino acids 121-190), respectively. For

pRS315-MMM1-GFP, a BamHI-XmaI fragment encoding the 0.52 kb MMM1 5’-UTR and

Mmm1p (amino acids 1-426), XmaI-EcoRV fragment encoding HA plus GFP, and an EcoRV-

XhoI fragment encoding the 0.45 kb MMM1 3’-UTR were generated by PCR and cloned into the

BamHI-XhoI sites of pRS315. For pRS315-mmm1 (2-75)-GFP and pRS315-mmm1 (2-90)-


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GFP, the 1.1 kb BamHI-SalI fragment of pRS315-MMM1-GFP was replaced by a 0.87 kb

BamHI-SalI fragment from pRS315-mmm1 (2-75)-His and a 0.82 kb BamHI-SalI fragment

from pRS315-mmm1 (2-90)-His, respectively. For pRS315-T20(TM)-mmm1(C)-His, a BamHI-

SpeI fragment encoding the 0.52 kb MMM1 5’-UTR plus ATG, a PCR-generated NheI-SpeI

fragment encoding Tom20p (amino acids 2-50), a SpeI-XhoI fragment encoding Mmm1p (amino

acids 121-426) plus 8xHis, and the 0.45 kb MMM1 3’-UTR were cloned into the BamHI-XhoI

sites of the pRS315 variant that lacks an SpeI site in the MCS. For pRS315-T20(TM)-mmm1(C)-

GFP, the 1.1 kb BamHI-SalI fragment of pRS315-MMM1-GFP was replaced by a 0.88 kb

BamHI-SalI fragment from pRS315-T20(TM)-mmm1(C)-His. For pRS315-T20(TM)-GFP, the

1.4 kb SpeI-XhoI fragment of pRS315-T20(TM)-mmm1(C)-His was replaced by a PCR-

generated SpeI-NcoI fragment encoding GFP (amino acids 2-56) and a 1.0 kb NcoI-XhoI

fragment from pRS315-MMM1-GFP. For pRS315-NcM(TM)-mmm1(C)-His, the 0.67 kb

BamHI-SpeI fragment of pRS315-T20(TM)-mmm1(C)-His was replaced by a PCR-generated

BamHI-NheI fragment encoding the 0.52 kb MMM1 5’-UTR plus ATG and N. crassa MMM1

(amino acids 2-39). For pRS315-NcM(TM)-mmm1(C)-GFP, the 1.1 kb BamHI-SalI fragment of

pRS315-MMM1-GFP was replaced by an 0.85 kb BamHI-SalI fragment from pRS315-

NcM(TM)-mmm1(C)-His. For pRS425-MMM1-GFP, a 3.0 kb BamHI-XhoI fragment from

pRS315-MMM1-GFP was cloned into the BamHI-XhoI sites of pRS425 (American Type Culture

Collection). For pRS425-mmm1(1-135)-GFP, pRS425-mmm1(1-180)-GFP, pRS425-mmm1(1-

275)-GFP, and pRS425-mmm1(1-365)-GFP, the 1.8 kb BamHI-XmaI fragment of pRS425-

MMM1-GFP was replaced by PCR-generated BamHI-XmaI fragments encoding the 0.52 kb

MMM1 5’-UTR plus Mmm1p (amino acids 1-135), (amino acids 1-180), (amino acids 1-275),

and (amino acids 1-365), respectively. For pRS425-mmm1(1-390)-GFP and pRS425-mmm1(1-


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415)-GFP, the 0.71 kb SalI-XmaI fragment of pRS425-MMM1-GFP was replaced by a PCR-

generated SalI-XmaI fragments encoding Mmm1p (amino acids 191-390) and (amino acids 191-

415), respectively. For pRS425-mmm1 (2-120)-GFP, the 1.1 kb BamHI-SalI fragment of

pRS425-MMM1-GFP was replaced by a 0.74 kb BamHI-SalI fragment from pRS315-mmm1 (2-

120)-His. For pRS315-TCS-MMM1-His and pRS315-TCS-MMM1-GFP, the 0.58 kb SpeI-SalI

fragment of pRS315-MMM1-His and pRS315-MMM1-GFP, respectively, was replaced by a

PCR-generated SpeI-NcoI fragment encoding 3xMyc, an NcoI-BglII fragment encoding TCS

(TEV cleavage site: ENLYFQG) plus 3xHA, and a BglII-SalI fragment encoding Mmm1p

(amino acids 2-190). For pRS315-TCSmut-MMM1-His, the 0.12 kb NcoI-BglII fragment of

pRS315-TCS-MMM1-His was replaced by a PCR-generated NcoI-BglII fragment encoding the

mutated TCS (ANLDFRP) plus 3xHA. For pYX142-TCS-NcMMM1, an EcoRI-SpeI fragment

encoding 3xMyc-TCS-3xHA and a SpeI-EcoRV fragment encoding N. crassa MMM1 (amino

acids 2-415) were generated by PCR and cloned into the EcoRI-EcoRV sites of pYX142, a low-

copy yeast expression vector with the strong constitutive TPI1 promoter (Novagen). For

pYX142-TCS-MMM1-His, the 1.4 kb NcoI-EcoRV fragment of pYX142-TCS-NcMMM1 was

replaced by a 0.69 kb NcoI-SalI fragment from pRS315-TCS-MMM1-His, and a 0.73 kb SalI-

EcoRV fragment from pRS416-MMM1-His. For pYX142-Su9-TCS-GFP, a SpeI-BglII fragment

encoding 3xHA plus TCS was generated by PCR, and the SpeI site was converted to blunt ends

using Mung Bean Nuclease. The fragment was cloned into the BamHI-BglII sites of pYX142-

mtGFP (the BamHI site was converted to blunt ends using DNA polymerase I, Large Fragment)

(37). For pYX142-b2-TCS-GFP, the 0.21 kb EcoRI-BglII fragment of pYX142-mtGFP was

replaced by a PCR-generated EcoRI-NheI fragment encoding Cyb2p (amino acids 1-220) and a

SpeI-BglII fragment encoding 3xHA plus TCS. For pRS416GAL1-b2-TEV, a PCR-generated


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NheI-BglII fragment encoding Cyb2p (amino acids 1-220) and a BamHI-XhoI fragment from

pWP1039 (provided by Will Prinz) encoding the TEV (tobacco etch virus) protease were cloned

into the XbaI-XhoI sites of pRS416GAL1 (American Type Culture Collection). All clones used

in this study were verified by DNA sequencing.

Site-specific TEV Cleavage Assay—Yeast cells harboring two plasmids encoding a TEV

protease and a substrate protein were pregrown to an OD600 of 1.5-3 in SD media. To induce TEV

protease expression, cells were washed three times with 1 ml of SGalR media, resuspended to an

OD600 of 0.4-1, and grown to an OD600 of 1-2 in SGalR media. 2 OD600 units of cells were

collected 3 h (for Su9-TCS-GFP), 6 h (for b2-TCS-GFP), or 16 h (for TCS-Mmm1p-His, TCSmut-

Mmm1p-His, and TCS-Mmm1p-GFP) after induction, resuspended in 1 ml of H2O, and lysed by

adding 160 µl of 1.85 M NaOH, 7.4% 2-mercaptoethanol on ice for 10 min. The protein extracts

were precipitated with 6% trichloroacetic acid on ice for 10 min. The precipitates were rinsed

with 1ml of 1M Tris base, resuspended in 60 µl of SDS gel-loading buffer, and boiled for 3 min.

15 µl of each sample was analyzed by SDS-PAGE and Western blotting.

Protease Protection Assay—Yeast cells expressing TCS-Mmm1p-His or TCS-Mmm1-GFP

were grown in SR media, and the mitochondria were isolated as previously described (38),

except that oxalyticase (Enzogenetics) was used for spheroplast formation (6 x 104 U in 30 ml

suspension containing 2 x 103 OD600 units of cells). Mitochondria (200 µg of protein) were

resuspended in 1 ml of HS buffer (20 mM HEPES-KOH, pH 7.4, 0.6 M sorbitol), H buffer (20

mM HEPES-KOH, pH 7.4), or HT buffer (20 mM HEPES-KOH, pH 7.4, 1% Triton X-100). If

indicated, the samples were treated with proteinase K at a final concentration of 200 µg/ml, and


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kept on ice for 30 min. The reaction was stopped by the addition of 2 mM phenylmethylsulfonyl

fluoride (PMSF) on ice for 5 min. For the samples in HS and H buffers, centrifugation was

performed at 16,000g for 10 min at 4°C. The pellet was washed with 1 ml of HS and H buffers

containing 1 mM PMSF, collected by centrifugation at 16,000g for 5 min at 4°C, and

resuspended in SDS gel-loading buffer. The sample in HT buffer was directly added to SDS gel-

loading buffer. The samples corresponding to 30 µg of mitochondria were boiled for 3 min, and

analyzed by SDS-PAGE and Western blotting.

Immunoblot Analysis—Western blots were decorated with mouse monoclonal antibodies

generated against HA (1:1,000; University of Utah Core Facility), GFP (1:1000; Covance), or 3-

PGK (1:2,000; Molecular Probes), and rabbit antisera raised against the N-terminal domain of

Fzo1p (1:2,000; Shaw laboratory), Cyb2p (1:2,000; Carla Koehler), or Ssc1p (1:1,000; Elizabeth

Craig) in TBS buffer containing 5% non-fat dry milk for 1 h, and washed three times with TBS

buffer for 10 min. The blots were decorated with the goat anti-mouse or -rabbit HRP conjugate

(1:8,000; Sigma) in TBS buffer containing 5% non-fat dry milk for 1 h, and washed three times

with TBS buffer for 10 min. Proteins were detected using ECL Plus and Hyperfilm ECL

(Amersham). All steps were performed at room temperature.

Microscopy and Imaging—Cells were visualized by a Zeiss Axioplan 2 Imaging microscope

(Carl Zeiss) equipped with differential interference contrast (DIC) optics, epifluorescence

capabilities, and a Zeiss Plan-Apochromat 100x (N.A. 1.4) objective. Filter sets (excitation /

beamsplitter / emission) for GFP and RFP fluorescence were BP 470 ± 20 / FT 495 / LP 525 ±

25, and BP 546 ± 6 / FT 560 / BP 575-640, respectively. Images were captured using a Zeiss


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AxioCam Mm monochrome digital camera and Zeiss AxioVision 3.1 software, and assembled

using Adobe Photoshop (Adobe Systems). Plasmids encoding organelle marker proteins, and a

staining probe used in this study were pVT100U-mtGFP (37), pRS424ADH-mtRFP (Shaw

laboratory), and MitoFluor Red 589 (Molecular Probes) for mitochondria, and pJK59 (Sec63p-

GFP) (39) for the endoplasmic reticulum.

RESULTS

The N-terminus of Mmm1p Is Exposed to the Mitochondrial Matrix—To investigate whether

the N-terminus of Mmm1p resides in the mitochondrial matrix or the IMS, we performed an in

vivo site-specific cleavage assay using Tobacco Etch Virus (TEV) protease (40) targeted either to

the matrix or the IMS. This protease recognizes the seven amino acid consensus sequence,

EXXYXQ(S/G). Cleavage occurs between the conserved glutamine and serine or glycine

residues (41). In vivo processing by TEV protease in budding yeast has been applied successfully

to examine localization and topology of peroxisomal and mitochondrial proteins tagged with

TEV cleavage sites (TCS) (42,43).

We constructed plasmids encoding TEV proteases fused to the C-termini of different

mitochondrial presequences including, Su9 (amino acids 1-69) from ATPase subunit 9 of N.

crassa, and b2 (amino acids 1-220) from cytochrome b2 of S. cerevisiae (Su9-TEV and b2-TEV,

respectively). Su9 and b2 are well-characterized presequences that specifically target various

passenger polypeptides, including TEV protease, to the matrix and the IMS, respectively

(44,37,43) (J. W. Thatcher and J. M. Shaw, unpublished data). To determine the location of the

N-terminus, Mmm1p-His was fused to the C-terminus of a wildtype TCS (TCS-Mmm1p-His) or

a mutated TCS (TCSmut-Mmm1p-His) sequence (Fig. 1A). In addition, GFP was fused to the C-


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terminus of TCS-Mmm1p (TCS-Mmm1p-GFP) (Fig. 1A). Each construct also contained a 1x-3x

HA epitope tag, allowing recognition by immunoblot analysis. The TCS-Mmm1p-His, TCSmut-

Mmm1p-His, and TCS-Mmm1p-GFP constructs were fully functional and restored normal

mitochondrial networks and glycerol growth when expressed in mmm1 cells (data not shown).

In addition, TCS-Mmm1p-GFP formed mitochondrial foci (data not shown).

Processing of the TCS-tagged Mmm1p variants by TEV protease is predicted to generate

proteins reduced in size by ~5 kDa that can be detected with anti-HA antibodies. When TCS-

Mmm1p-His was coexpressed with Su9-TEV, low levels of the ~5 kDa shorter form were

detected (Fig. 1B, lane 2, arrow; for each substrate, Su9-TEV and b2-TEV were expressed under

identical conditions and for identical times). In contrast, very little of the ~5 kDa shorter form of

TCS-Mmm1p-His was produced by the coexpression of b2-TEV (Fig. 1B, lane 4). In time-course

experiments, the amount of cleavage product produced by expression of Su9-TEV protease was

consistently larger than that produced by b2-TEV expression (data not shown). The cleavage of

TCS-Mmm1p-His by Su9-TEV occurred within the TCS, since Su9-TEV did not generate the ~5

kDa shorter form of the TCSmut-Mmm1p-His substrate (Fig. 1B, lane 2). Moreover, specific Su9-

TEV dependent processing was observed in cells expressing TCS-Mmm1p-GFP (Fig. 1B, lane 2,

open arrowhead). Although the processing of TCS-Mmm1p-His and TCS-Mmm1p-GFP was not

complete, additional control experiments indicated that it was specific to Su9-TEV. In particular,

the substrates Su9-TCS-GFP and b2-TCS-GFP were processed only by Su9-TEV and b2-TEV,

respectively (Fig1B, lane2, closed arrowhead and 4, closed circle). The latter results demonstrate

that TEV proteases are active in the matrix and IMS, and that the processing occurs only when

both a TEV protease and a substrate are localized in the same mitochondrial subcompartment.

Together, these results suggest that the N-terminus of Mmm1p is exposed in the matrix.


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To further examine the topology of Mmm1p, we carried out a conventional protease

protection assay in vitro. Mitochondria were isolated from mmm1 cells expressing TCS-

Mmm1p-His or TCS-Mmm1p-GFP, and incubated in the presence or absence of proteinase K

(PK) with no treatment (to keep the organelle intact), osmotic shock treatment (to expose the

IMS), or Triton X-100 treatment (to solubilize the organelle). As shown in Figure1C, lanes 1 and

5, anti-HA antibodies decorated the full-length ~80 kDa TCS-Mmm1p-His and ~107 kDa TCS-

Mmm1p-GFP polypeptides in mitochondrial samples that were not treated with protease. In

contrast, when intact mitochondria were exposed to PK (200 µg/ml), a ~40 kDa proteolytic

fragment was detected (Fig. 1C, lanes 2 and 6). The apparent molecular weight of the protected

polypeptide was larger than predicted for this substrate (a cleavage product consisting of 3xMyc-

TCS-3xHA plus the first 120 amino acid residues of Mmm1p should migrate at ~22 kDa). This

size increase was not due to incomplete proteolysis, as treatment with 1 mg/ml PK also generated

a ~40 kDa form (data not shown). During the course of this study, we noticed that all variants of

Mmm1p migrated slower (appeared larger) on SDS gels than predicted. The reason for this

altered migration is not known. Nevertheless, when osmotically shocked mitochondria

containing TCS-Mmm1p-His or TCS-Mmm1p-GFP were treated with PK, the ~40 kDa clipped

form was not degraded further, even though the OM was clearly ruptured, allowing the IMS

protein Cyb2 to be digested (Fig. 1C, lane 3 and 7). In contrast, the protected ~40 kDa form was

completely degraded and could not be detected in PK-treated samples containing Triton X-100

(Fig. 1C, lane 4 and 8). Under this condition, both mitochondrial membranes were lysed as

assessed by degradation of the matrix protein Ssc1. Since the HA tag recognized in these

experiments resides near the N-termini of the substrates, these results indicate that the N-termini

of TCS-Mmm1p-His and TCS-Mmm1p-GFP can only be digested when PK has access to


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proteins in the matrix. Similar results were obtained when experiments were performed using a

different protease, trypsin (data not shown). Based on the combined results, we conclude that

Mmm1p has the topology of Nmatrix-Ccytoplasm, spanning the outer and inner mitochondrial

membranes.

The N-terminal Extension of Mmm1p Is Not Required for Maintenance of Mitochondrial

Morphology and MtDNA—To test whether the N-terminal extension of Mmm1p is critical for

mitochondrial morphology and/or mtDNA maintenance, we constructed plasmids encoding

Mmm1 mutant proteins that lack amino acid residues 2-90 and 2-120 (Mmm1∆(2-90)p-His and

Mmm1∆(2-120)p-His, respectively) (Fig. 2A). Mmm1∆(2-90)p-His lacks nearly the entire N-terminal

extension in front of the predicted TM segment (amino acids 92-116) (27). Both the N-terminal

extension and the TM segment are deleted in Mmm1∆(2-120)p-His. When expressed in mmm1

cells, Mmm1∆(2-90)p-His restored normal mitochondrial tubular networks that were

indistinguishable from those in mmm1 cells expressing full-length Mmm1p-His (Fig. 2B and

Table I). In contrast, mmm1 cells expressing Mmm1∆(2-120)p-His exhibited the large spherical

mitochondria typically seen in mmm1 cells containing an empty plasmid (Fig. 2B and Table I).

Although Mmm1p-His and Mmm1∆(2-90)p-His restored glycerol growth when shuffled into

mmm1 cells, mmm1 cells expressing Mmm1∆(2-120)p-His or containing an empty plasmid did

not grow on glycerol medium (Fig. 2C). No glycerol growth defect was observed in mmm1

cells expressing Mmm1∆(2-90)p-His at 37°C (data not shown). Consistent with this growth pattern,

the morphology and number of DAPI-stained mtDNA nucleoids in mmm1 cells expressing

Mmm1p-His or Mmm1∆(2-90)p-His were similar, while mmm1 cells expressing Mmm1∆(2-120)p-

His or containing an empty plasmid lacked such structures (data not shown). The failure of


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Mmm1∆(2-120)p-His to complement mutant phenotypes in mmm1 cells is likely due to the

absence of the TM segment, which is required for mitochondrial membrane insertion (see Fig. 6).

Our data suggest that the N-terminal extension of Mmm1p is dispensable for the maintenance of

normal mitochondrial tubular networks and mtDNA.

Yeast mmm1 Cells Expressing NcMMM1 Can Maintain Normal Mitochondrial Tubular

Networks and MtDNA—Although S. cerevisiae Mmm1p is fairly similar to the N. crassa

homologue NcMMM1 (30% identity and 54% similarity), there is a significant difference

between the two proteins at the N-termini. In particular, Mmm1p has ~90 amino acid residues N-

terminal to the predicted TM segment, while NcMMM1 has ~10 amino acid residues (32). The

fact that Mmm1∆(2-90)p-His is as functional as Mmm1p-His prompted us to test whether

NcMMM1 can substitute for the yeast homologue. We constructed a yeast expression vector

encoding NcMMM1 fused to the C-terminus of a TCS (TCS-NcMMM1). The same vector was

used for the expression of TCS-Mmm1p-His as a control. When expressed in mmm1 cells,

TCS-NcMMM1 restored normal mitochondrial tubular networks that were virtually identical to

those in mmm1 cells expressing TCS-Mmm1p-His (Fig. 3A and Table I). In addition, the

glycerol growth defect of mmm1 cells was suppressed by the expression of TCS-NcMMM1 or

TCS-Mmm1p-His at 30°C (Fig. 3B) and 37°C (data not shown). DAPI-staining confirmed that

normal mtDNA nucleoids were present in mmm1 cells expressing TCS-NcMMM1 or TCS-

Mmm1p-His (data not shown). In vivo TEV cleavage and in vitro protease protection assays

suggest that the N-terminus of NcMMM1 is also exposed to the matrix of yeast mitochondria (N.

Kondo-Okamoto, unpublished data). Together, these results demonstrate that, despite the lack of

an N-terminal extension, NcMMM1 can perform the function of Mmm1p in yeast.


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Deletion of the Mmm1p N-terminal Extension Does Not Affect Foci Formation but Reduces

the Steady-state Level of Mmm1p—To examine whether the N-terminal extension of Mmm1p is

required for foci formation, we constructed plasmids encoding Mmm1p-GFP mutant proteins

lacking the amino acid residues 2-75 and 2-90 (Mmm1∆(2-75)p-GFP and Mmm1∆(2-90)p-GFP,

respectively) (Fig. 4A). When expressed in mmm1 cells, both Mmm1∆(2-75)p-GFP and Mmm1∆(2-

90)p-GFP formed discrete foci on mitochondrial tubules identical to those observed in mmm1

cells expressing Mmm1p-GFP (Fig. 4B) (30). The number of cells exhibiting these Mmm1p foci,

the number of foci per cell, and the fluorescence intensity of the foci were similar for all three

constructs, Mmm1∆(2-75)p-GFP, Mmm1∆(2-90)p-GFP, and Mmm1p-GFP (data not shown).

Moreover, the morphology of restored mitochondrial networks was identical in mmm1 cells

expressing Mmm1∆(2-75)p-GFP and Mmm1∆(2-90)p-GFP, and Mmm1p-GFP (Fig. 4B) (30). These

observations suggest that the N-terminal extension of Mmm1p is dispensable for foci formation.

Surprisingly, we found that the steady-state level of Mmm1∆(2-90)p-GFP was four- to six-fold

lower than that of Mmm1p-GFP and Mmm1∆(2-75)p-GFP (Fig. 4C). This observation indicates that

amino acid residues 75-90 somehow affect expression or turnover of the Mmm1 mRNA or

protein. Since mutant fusion proteins lacking the N-terminal extension function as well as the

wildtype fusion protein and produce similar numbers of Mmm1p foci, these data suggest that the

steady-state level of Mmm1p expressed in wildtype cells is not limiting.

The TM Segment of Mmm1p Is Essential for Foci Formation—To investigate the role of the

Mmm1p TM segment in foci formation, we constructed plasmids encoding chimeric proteins

containing the TM segment of Tom20p or NcMMM1, and the large C-terminal domain of


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Mmm1p-His (T20(TM)-Mmm1(C)p-His and NcM(TM)-Mmm1(C)p-His, respectively) (Fig. 5A). For

localization studies, GFP was fused at the C-terminus of T20(TM)-Mmm1(C)p and NcM(TM)-

Mmm1(C)p (T20(TM)-Mmm1(C)p-GFP and NcM(TM)-Mmm1(C)p-GFP, respectively) (Fig. 5A).

Tom20p contains a single TM segment at the N-terminus embedded in the outer mitochondrial

membrane, and has the topology of Nin-Ccytoplasm (45). A previous study showed that the human

Tom20 TM segment plus some flanking residues fused at the N-terminus of GFP can be targeted

and inserted into the OM in humans (46).

When expressed in wildtype cells, GFP fused to the C-terminus of the Tom20p TM segment

(T20(TM)-GFP, Fig. 5A) colocalized with mitochondria and was evenly distributed throughout the

tubular network (Fig. 5B). Similarly, T20(TM)-Mmm1(C)p-GFP localized throughout the

mitochondrial network without forming visible foci (Fig. 5B). In contrast, NcM(TM)-Mmm1(C)p-

GFP formed foci that colocalized with mitochondrial tubules (Fig. 5B). These results indicate

that the TM segment of Mmm1p is essential for foci formation, and can be replaced with that of

NcMMM1. Interestingly, T20(TM)-Mmm1(C)p-His did not restore normal mitochondrial tubular

networks and glycerol growth in mmm1 cells, whereas NcM(TM)-Mmm1(C)p-His suppressed

these defects in the mutant (Fig. 5C, D and Table I). These data, together with the observation

that the N-terminus of NcMMM1 is exposed to the matrix of yeast mitochondria (N. Kondo-

Okamoto, unpublished data), suggest that the double membrane-spanning topology is crucial for

Mmm1p to localize as discrete foci where the protein performs its function.

The C-terminal Domain of Mmm1p Is Indispensable for Targeting to Mitochondria—To

define the region required for targeting Mmm1p to mitochondria, we expressed plasmids

encoding GFP fused to the C-terminus of various Mmm1p deletion mutants in wildtype cells


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(Fig. 6A). Surprisingly, Mmm1(1-135)p-GFP was targeted to perinuclear structures similar to those

labeled with the endoplasmic reticulum (ER) marker Sec63p-GFP (39) (Fig. 6B). Since we

already showed that removal of the N-terminus does not interfere with Mmm1p targeting or foci

formation, this finding indicates that the TM segment of Mmm1p does not contain a complete

signal for targeting to mitochondria. Additional studies established that Mmm1(1-180)p-GFP and

Mmm1(1-275)p-GFP were also targeted to the ER, while Mmm1(1-365)p-GFP and Mmm1(1-390)p-GFP

were targeted to both the ER and cytoplasm (Fig. 6A). In contrast, Mmm1(1-415)p-GFP formed

discrete foci that were colocalized with the mitochondrial network (Fig. 6B), and restored normal

mitochondrial morphology and glycerol growth in mmm1 cells (data not shown). These results

indicate that the last 11 amino acids of Mmm1p are not required for mitochondrial targeting and

foci formation. Mmm1∆(2-120)p-GFP, a mutant lacking the TM segment, was targeted to the

cytoplasm (Fig. 6B). Collectively, these observations demonstrate that, together with the TM

segment, the C-terminal domain plays an important role in targeting Mmm1p to mitochondria.

DISCUSSION

Double Membrane-spanning Topology of Mmm1p—Previous studies suggested that: 1)

Mmm1p spans the OM with its C-terminus exposed to the cytoplasm (27), and 2) Mmm1p forms

discrete foci at membrane contact sites, adjacent to mtDNA nucleoids in the matrix (30).

Although the latter finding raised the possibility that the N-terminus of Mmm1p spans the IM,

the submitochondrial location of Mmm1p’s N-terminus has not been reported (30). In this study,

we used in vivo site-specific cleavage and in vitro protease protection assays to demonstrate that

the N-terminus of Mmm1p is exposed to the mitochondrial matrix. Together, these findings

establish that Mmm1p spans both mitochondrial membranes with the topology of Nmatrix-Ccytoplasm.


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Tim23p is the only other protein reported to span both mitochondrial membranes in yeast

(47). This protein contains an N-terminal domain anchored in the OM, a middle (hydrophilic)

domain exposed to the IMS, and four, C-terminal TM helices embedded in the IM (47). Unlike

Tim23p, Mmm1p contains only one predicted TM segment and is unlikely to contain a stretch

exposed to the IMS. This interpretation is supported by our observation that the N-terminal

extension of Mmm1p was not degraded further by externally added protease even when the OM

was ruptured by osmotic shock (Fig. 1C).

In the absence of a second TM segment, at least three different models could account for the

double membrane-spanning topology of Mmm1p. First, Mmm1p might span both mitochondrial

membranes once via its single TM segment, as depicted in Fig. 7A. This scenario is problematic,

if the TM helix of Mmm1p is only ~25 amino acid residues in length (amino acids 92-116), as

previously predicted (Kyte-Doolittle hydrophobicity prediction) (27). Unless this region adopts

an unconventional structure (e.g. a dramatically extended helix), additional amino acids flanking

the predicted TM helix would be required to span two lipid bilayers. In fact, other prediction

programs including SOSUI (amino acids 102-123), HMMTOP (amino acids 101-120) and

TMHMM (amino acids 100-122), predict slightly different TM helices within the Mmm1 protein

(ExPASy Molecular Biology Server). When the results of all four prediction programs are

combined, an Mmm1p TM segment of 32 residues (amino acids 92-123) results. If a TM

segment of this length exists in Mmm1p, it might be sufficient to span both mitochondrial

membranes. Second, the TM segment of Mmm1p could be embedded in the OM and the N-

terminal extension could span the IM through an aqueous pore formed by other members of a

protein complex (Fig. 7B). Third, the C-terminal flanking region of the Mmm1p TM segment

could span the OM through an aqueous pore formed by proteins in the OM (Fig. 7C). Further


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studies are required to define the length, position, structure and environment of Mmm1p’s

double membrane-spanning domain.

A recent study indicates that two additional OM proteins, Mdm10p and Mdm12p, form

mitochondrial foci adjacent to mtDNA nucleoids (48). Like the mmm1 strain, cells lacking

Mdm10p and Mdm12p contain spherical mitochondria and lose mtDNA nucleoids (25,26). In

addition, all three proteins behave as if they form a complex in immune precipitation

experiments (48), and localization of Mmm1p to mitochondrial foci does not occur in the

absence of Mdm10p and Mdm12p (48) (N. Kondo-Okamoto, unpublished data). Based on these

observations, it is likely that Mdm10p and Mdm12p interact with Mmm1p, forming a complex at

the membrane contact site. The topology of Mdm10p and Mdm12p with respect to both

mitochondrial membranes, and the domains of those proteins required for interaction with

Mmm1p are important issues to be addressed in the future.

Role of the Mmm1p TM Segment in Foci Formation and Generation of Contact Sites—Our

finding that the double membrane-spanning topology of Mmm1p is critical for foci formation

(Fig. 5) supports the notion that Mmm1p functions at contact sites between the inner and outer

mitochondrial membranes. Although we cannot rule out the possibility that a subgroup of

Mmm1p molecules is distributed throughout mitochondria in a single membrane-spanning

fashion, the results presented here suggest that the membrane contact site is the place where

Mmm1p forms a functional complex. Despite three decades of research on mitochondrial

structure and protein import, proteins that direct the formation of these morphologically defined

membrane contact sites have proven difficult to identify (49). Here we demonstrate that the

double membrane-spanning protein Mmm1 serves as a marker of this intramitochondrial


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structure. Is Mmm1p a structural determinant of the mitochondrial membrane contact site,

attaching the OM to the IM? Based on the observation that the IM is strikingly disorganized in

mmm1 cells, a role for Mmm1p in forming this structure has been proposed (30). However, the

disorganized IM observed in mmm1 mutants could also be a secondary consequence of

converting mitochondrial membranes from tubular networks to spherical compartments.

Moreover, it has not been firmly established whether the membrane contact sites are lost and/or

destabilized in mitochondria lacking Mmm1p. High-resolution electron microscopy and 3D

tomography of mmm1 mitochondria will help to address this issue.

Role of the Mmm1p N-terminal Extension in MtDNA Nucleoid Maintenance—The unique

Nmatrix-Ccytoplasm topology of Mmm1p raised the possibility that this is a bifunctional protein with

two topologically distinct domains, an N-terminal extension for mtDNA nucleoid maintenance

and a C-terminal domain for mitochondrial morphology maintenance. This hypothesis predicts

that deletion of the N-terminal extension will lead to mtDNA loss but will not affect

mitochondrial morphology. However, data presented here indicate that the N-terminal extension

of Mmm1p is not required for maintenance of normal tubular networks or mtDNA nucleoids

(Fig. 2). Thus, it is unlikely that Mmm1p directly anchors mtDNA nucleoids to the matrix side of

the IM. This interpretation is supported by our observation that, despite the lack of an N-terminal

extension, the N. crassa MMM1 homologue is fully functional in budding yeast (Fig. 3).

How is Mmm1p involved in maintenance of mtDNA? We cannot exclude the possibility that

Mmm1p functions indirectly to attach mtDNA nucleoids to the IM. For example, Mmm1p may

interact with one or more proteins whose matrix-exposed domains directly bind mtDNA

nucleoids. In the absence of functional Mmm1p, such protein(s) might not target, assemble or


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function properly to anchor mtDNA nucleoids at the membrane contact site, ultimately resulting

in mtDNA loss. Alternatively, unknown factor(s) might anchor mtDNA nucleoids to the

membrane contact site independently of Mmm1p. In this scenario, mtDNA nucleoid aggregation

and the subsequent mtDNA loss phenotypes of mmm1 mutants (30) may be a secondary

consequence of mitochondrial morphology defects that grossly alter IM structure.

Role of Mmm1p in Cytoskeletal Attachment—Although a role for Mmm1p in cytoskeletal

attachment has been proposed, two lines of evidence suggest that this protein does not directly

mediate interactions between mitochondria and the cytoskeleton. First, in N. crassa,

microtubules are the cytoskeletal elements utilized for mitochondrial morphology, distribution

and inheritance (31). However, in vitro studies showed that NcMMM1-depleted mitochondria

still bind to microtubules (33), indicating that NcMMM1 is not essential for microtubule-

dependent mitochondrial behavior. Second, we show here that NcMMM1 can substitute for

Mmm1p in S. cerevisiae (Fig. 3). In contrast to N. crassa, S. cerevisiae utilizes the actin

cytoskeleton for mitochondrial attachment and movement (5,50). Thus, the primary function of

Mmm1p has apparently been conserved between S. cerevisiae and N. crassa, independent of the

cytoskeletal system used by the cell to regulate mitochondrial dynamics. Based on these results,

it seems unlikely that the Mmm1 protein acts as a direct molecular bridge between mitochondria

and cytoskeletal elements to maintain tubular networks.

How, then, can we reconcile the observation that S. cerevisiae mitochondria bind actin

filaments in an Mmm1p-dependent fashion in vitro (28)? One possibility is that S. cerevisiae and

N. crassa Mmm1 proteins bind molecular adaptors that bridge interactions between mitochondria

and actin filaments in both cell types. Loss of actin interactions would have a severe affect in


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budding yeast, since actin filaments and cables are the major cytoskeletal system found in the

cytoplasm. In contrast, both actin and microtubules are found as prominent cytoplasmic

structures in N. crassa. Since N. crassa relies most heavily on microtubules for mitochondrial

dynamics and presumably has additional proteins that mediate microtubule attachment, loss of

Mmm1p actin-binding activity produces only subtle (if any) defects. Identifying the complete

set of cytoskeleton-binding and motor proteins that interact with mitochondria in both organisms

will help to clarify this discrepancy.

The Mmm1p Cytoplasmic Domain Is Required for Mitochondrial Targeting—To our surprise,

we found that Mmm1p mutant proteins lacking the cytoplasmic C-terminal domain were targeted

to the ER rather than mitochondria (Fig. 6). This result indicates that all or a part of the C-

terminal domain serves as a “subsignal” for mitochondrial targeting of Mmm1p. To date, only

one other protein is known to require cytoplasmic signals for mitochondrial targeting. An alpha

helix at the N-terminus of the single TM protein Bax was recently shown to contribute

significantly to mitochondrial targeting (51). These cytoplasmic “subsignals” may interact with

receptor proteins or specific recruiting factors that facilitate faithful targeting.

Why are Mmm1p mutants lacking the C-terminal domain targeted to the ER? This

phenomenon may be due to the hydrophobicity and/or length of the membrane-spanning domain.

For example, human Tom20 variants with increased hydrophobicity and length in the TM

segment are targeted to ER/Golgi compartments (46). Similarly, increasing the TM domain

hydrophobicity of another outer mitochondrial membrane protein, Fis1, also results in ER

targeting (52). In the absence of cytoplasmic C-terminal sequences, the excess hydrophobicity

and/or length of the Mmm1p TM segment may define a default signal for ER targeting.


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What are the proteins that interact with the C-terminal domain of Mmm1p and recruit

Mmm1p to mitochondria? As described above, recent studies suggest that at least two other

proteins, Mdm10p and Mdm12p, are required to target Mmm1p to mitochondria (48) (N. Kondo-

Okamoto, unpublished observations). In addition to components of the OM protein import

machinery, other, as yet unidentified, proteins may play a role in targeting Mmm1p to

mitochondria. Studying how Mmm1p is targeted to mitochondria, inserted into the outer and

inner membranes, and assembled into a complex at the membrane contact site will help us to

understand the molecular function of Mmm1p and the mechanisms and pathways underlying

protein transport to a distinct intramitochondrial structure, the membrane contact site.

Acknowledgments—We thank Benedikt Westermann (University of Munich, Munich,

Germany) for plasmids pVT100U-mtGFP, pYX142-mtGFP, and the cDNA encoding NcMMM1,

Will Prinz (National Institute of Health, Bethesda, MD) for plasmids pJK59 and pWP1039, Carla

Koehler (University of California Los Angeles, Los Angeles, CA) for anti-Cyb2p antisera,

Elizabeth Craig (University of Wisconsin, Madison, WI) for anti-Ssc1p antisera, and members of

the Shaw laboratory for stimulating discussions and careful review of the manuscript. N.K.-O.

and K.O. are deeply grateful to Walter Neupert (University of Munich, Munich, Germany) for

his generous support during the initial stage of this study.

This work was supported by National Institutes of Health Grant GM-53466 awarded to J.M.S

and a grant from the United Mitochondrial Disease Foundation awarded to K.O. The University

of Utah DNA and Peptide Facility is supported in part by a grant from the National Cancer

Institute (CA42014).


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FIGURE LEGENDS

Fig. 1. The N-terminus of Mmm1p is exposed to the mitochondrial matrix. A, schematic

representation of proteins used for in vivo site-specific cleavage and in vitro protease protection

assays. Mmm1p is 426 amino acids in length. The TM segment designated in Burgess et al.

(1994) is shown (amino acids 92-116). B, in vivo site-specific cleavage in cells harboring

plasmids that encode a TEV protease (top) and a substrate protein (left). The expression of TEV

proteases was repressed by dextrose (lanes 1 and 3) or induced by galactose (lanes 2 and 4).

Protein extracts from 0.5 OD600 units of cells were analyzed by immunostaining with antibodies

specific for HA (for TCS-Mmm1p-His, TCSmut-Mmm1p-His and TCS-Mmm1p-GFP expressed

in mmm1 cells) and GFP (for Su9-TCS-GFP and b2-TCS-GFP expressed in wildtype cells). The

asterisk indicates a form of Su9-TCS-GFP produced by the mitochondrial processing peptidase.

TEV protease cleavage products for each substrate are marked to the left of each panel by an

arrow, open arrowhead, closed arrowhead or closed circle. C, in vitro proteinase K (PK)

treatment of mitochondria isolated from mmm1 cells expressing TCS-Mmm1p-His or TCS-

Mmm1p-GFP. Mitochondria (30 µg of protein) were mock-treated (lanes 1 and 5), treated with

200 µg/ml PK under osmotic conditions (lanes 2 and 6) or subjected to osmotic shock (OS) in

the absence (lanes 3 and 7) or presence (lanes 4 and 8) of Triton X-100 (TX-100). Samples were

analyzed by immunostaining with antibodies specific for HA (for TCS-Mmm1p-His and TCS-

Mmm1p-GFP), Fzo1p (for the OM protein), Cyb2p (for the IMS protein) and Ssc1p (for the

matrix protein). Asterisks indicate forms produced by non-specific degradation.

Fig. 2. The N-terminal extension of Mmm1p is not essential for mitochondrial

morphology or mtDNA maintenance. A, schematic representation of Mmm1p deletion mutant


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proteins. B, mmm1 cells carrying plasmid pRS315 alone (no protein expressed) or encoding

Mmm1p-His, Mmm1∆(2-90)p-His, or Mmm1∆(2-120)p-His were transformed with pVT100U-mtGFP,

grown to log phase in SD media and observed by fluorescence microscopy to visualize

mitochondria. Bar, 5 µm. C, cells described in B (without pVT100U-mtGFP) were grown

overnight in SD media, spotted as two-fold serial dilutions, and incubated for three (SD plates)

or six (SGly plates) days.

Fig. 3. N. crassa MMM1 can substitute for Mmm1p in yeast. A, mmm1 cells carrying the

plasmid pYX142 alone (no protein expressed) or encoding TCS-Mmm1p-His, or TCS-

NcMMM1 were transformed with pVT100U-mtGFP, grown to log phase in SD media, and

observed by fluorescence microscopy to visualize mitochondria. Bar, 5 µm. B, cells described in

A (without pVT100U-mtGFP) were grown overnight in SD media, spotted as two-fold serial

dilutions, and incubated for three (SD plates) or six (SGly plates) days.

Fig. 4. Deletion of the Mmm1p N-terminal extension does not affect foci formation but

reduces steady-state protein levels. A, schematic representation of Mmm1p deletion variants

fused to the N-terminus of GFP. B, mmm1 cells carrying the plasmid pRS315 encoding

Mmm1p-GFP, Mmm1∆(2-75)p-GFP, or Mmm1∆(2-90)p-GFP were grown to log phase in SD media,

stained with MitoFluor Red 589, and observed by fluorescence microscopy. Bar, 5 µm. C,

protein extracts from 0.5 OD600 units of cells described in B were analyzed by immunostaining

with antibodies specific for GFP (for Mmm1p-GFP, Mmm1∆(2-75)p-GFP, and Mmm1∆(2-90)p-GFP)

and 3-PGK (as a cytoplasmic protein loading control).


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Fig. 5. The double membrane-spanning topology of Mmm1p is crucial for foci

formation. A, schematic representation of chimeric fusion proteins. B, wildtype cells carrying

the plasmid pRS315 encoding T20(TM)-GFP, T20(TM)-Mmm1(C)p-GFP, or NcM(TM)-Mmm1(C)p-GFP

were grown to log phase in SD media, stained with MitoFluor Red 589, and observed by

fluorescence microscopy. Bar, 5 µm. C, mmm1 cells carrying the plasmid pRS315 encoding

T20(TM)-Mmm1(C)p-His or NcM(TM)-Mmm1(C)p-His were transformed with pVT100U-mtGFP,

grown to log phase in SD media, and observed by fluorescence microscopy to visualize

mitochondria. Bar, 5 µm. D, mmm1 cells carrying the plasmid pRS315 alone (no protein

expressed) or encoding Mmm1p-His, T20(TM)-Mmm1(C)p-His or NcM(TM)-Mmm1(C)p-His were

grown overnight in SD media, spotted as two-fold serial dilutions, and incubated for three days

(SD plates) or six days (SGly plates).

Fig. 6. The C-terminal domain of Mmm1p plays an essential role in mitochondrial

targeting. A, schematic representation of Mmm1p deletion variants fused to the N-terminus of

GFP. Wildtype cells containing pRS425 encoding each GFP fusion protein or pJK59 encoding

the ER marker Sec63p-GFP were transformed with pRS424ADH-mtRFP, grown to log phase in

SD media and observed by fluorescence microscopy. Localization of these GFP fusion proteins is

summarized at the right. B, representative images of wildtype cells expressing Mmm1p-GFP,

Mmm1(1-135)p-GFP, Mmm1(1-415)p-GFP, Mmm1∆(2-120)p-GFP, or Sec63p-GFP. Bar, 5 µm.

Fig. 7. Schematic models for the double membrane-spanning topology of Mmm1p.

Asterisks mark hypothetical pore-forming proteins in the outer or inner membrane. OM, outer

membrane; IMS, intermembrane space; IM, inner membrane. See text for details.


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Table I. Mitochondrial Membrane Morphology in mmm1 Cells

Plasmid %Tubule %Intermediate* %Sphere

pRS315 5 30 65pRS315-MMM1-His 89 7 4pRS315-mmm1 (2-90)-His 94 4 2pRS315-mmm1 (2-120)-His 7 45 48

pRS315-T20(TM)-mmm1(C)-His 14 65 21pRS315-NcM(TM)-mmm1(C)-His 87 10 3

pYX142 12 41 47pYX142-TCS-MMM1-His 85 12 3pYX142-TCS-NcMMM1 96 2 2

Numbers are percent of cells grown to log phase in SD media at 30°C (n=200). Mitochondrial

morphology was visualized with matrix-targeted GFP, and scored in the three different categories

shown. *Intermediate denotes coexistence of tubules and spheres, or fragmented thick tubules.


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Noriko Kondo-Okamoto, Janet M. Shaw and Koji Okamotodistinct domains for targeting and foci formation

Mmm1p spans both the outer and inner mitochondrial membranes, and contains

published online September 12, 2003J. Biol. Chem.

10.1074/jbc.M308436200Access the most updated version of this article at doi:

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mmm1p spans both the outer and inner mitochondrial membranes

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