MICROSCOPY RESEARCH AND TECHNIQUE 51:573–583 (2000)
Yeast Mitochondrial Dynamics: Fusion, Division,
Segregation, and Shape
ROBERT E. JENSEN,* ALYSON E. AIKEN HOBBS, KARA L. CERVENY,
AND
HIROMI SESAKI
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
ABSTRACT
Mitochondria are essential organelles found in virtually all eukaryotic cells that
play key roles in a variety of cellular processes. Mitochondria show a striking heterogeneity in their
number, location, and shape in many different cell types. Although the dynamic nature of mitochondria has been known for decades, the molecules and mechanisms that mediate these processes
are largely unknown. Recently, several laboratories have isolated and analyzed mutants in the
yeast Saccharomyces cerevisiae defective in mitochondrial fusion and division, in the segregation of
mitochondria to daughter cells, and in the establishment and maintenance of mitochondrial shape.
These studies have identified several proteins that appear to mediate different aspects of mitochondrial morphogenesis. Although it is clear that many additional components have yet to be
identified, some of the newly discovered proteins raise intriguing possibilities for how the processes
of mitochondrial division, fusion, and segregation occur. Below we summarize our current understanding of the molecules known to be required for yeast mitochondrial dynamics. Microsc. Res.
Tech. 51:573–583, 2000. © 2000 Wiley-Liss, Inc.
INTRODUCTION
Mitochondria play important and fundamental roles
in ATP synthesis, ion homeostasis, lipid metabolism,
cell fate determination, apoptosis, and aging (Attardi
and Schatz, 1988; Green and Reed, 1998; Saraste,
1999; Tzagoloff, 1983; Wallace, 1999). To accommodate
such diverse functions, mitochondria often establish
specific numbers and locations, as well as specialized
shapes in different cell types (Bereiter-Hahn, 1990;
Bereiter-Hahn and Voth, 1994; Munn, 1974; Tandler
and Hoppel, 1972). For example, in muscle fibers, mitochondria make up more than 30% of the volume of
the cell, and are stacked into tubular arrays between
rows of actin-myosin bundles, facilitating the efficient
delivery of ATP. In cells like fibroblasts, mitochondria
make up only a small percentage of the cell volume,
and show a more dispersed distribution. Mitochondrial
shape also exhibits striking variations in different
cells, ranging from small, football-shaped organelles to
elongated, branched tubular networks.
The yeast Saccharomyces cerevisiae dramatically
regulates the shape, size, and number of its mitochondria during cell growth (Hermann and Shaw, 1998;
Yaffe, 1999). Under anaerobic conditions, mitochondria
form very small organelles called promitochondria
(Criddel and Schatz, 1969; Plattner and Schatz, 1969).
When yeast are grown aerobically, mitochondria become enlarged and elongated, and are positioned at the
cell periphery (Hoffman and Avers, 1973; Stevens,
1977, 1981). This location is proposed to place the mitochondria near the point of entry of oxygen, and the
elongated structure is thought to facilitate the rapid
conduction of ATP through the cell. The tubular-reticular structure of yeast mitochondria seen during vegetative growth (see Fig. 1) is typical of mitochondrial
structure in many different eukaryotic cells. In addition to their shape and size, the number of mitochondria in a yeast cell can also vary. During logarithmic
©
2000 WILEY-LISS, INC.
growth, yeast cells have from one to ten elongated
mitochondria (Hoffman and Avers, 1973; Stevens,
1977, 1981). The number of mitochondria per cell is not
absolutely constant since mitochondria are frequently
fusing and dividing (Bereiter-Hahn, 1990; BereiterHahn and Voth, 1994; Nunnari et al., 1997). Furthermore, when yeast cells enter stationary phase, the mitochondrial tubules fragment to form many small,
round mitochondria (Stevens, 1977, 1981).
The dynamic nature of mitochondria in eukaryotic
cells has been apparent since the turn of the century,
when live cells were first examined using the light
microscope (Lewis and Lewis, 1915). However, only
recently have any of the proteins that mediate mitochondrial morphogenesis been identified. Yeast molecular genetics has made key contributions to the identification of many of the molecules required for mitochondrial dynamics: the fusion and fission of mitochondria, the segregation of mitochondria to daughter
cells following cytokinesis, and the maintenance of mitochondrial shape.
ISOLATION OF YEAST MUTANTS
DEFECTIVE IN MITOCHONDRIAL DYNAMICS
To identify the proteins required for mitochondrial
dynamics, several laboratories have isolated mutants
in the yeast Saccharomyces cerevisiae that are defective in the shape, number, or segregation of mitochondria (Burgess et al., 1994; Hermann et al., 1997; McConnell et al., 1990; Meeusen et al., 1999). In the initial
genetic approaches, collections of temperature-sensitive yeast mutants were individually grown, stained
with mitochondrial-specific fluorescent dyes, and then
*Correspondence to: Robert E. Jensen, Dept. of Cell Biology and Anatomy,
Biophysics 100, The Johns Hopkins University School of Medicine, 725 N. Wolfe
St., Baltimore, MD 21205. E-mail: rjensen@jhmi.edu
Received 25 May 2000; accepted in revised form 20 July 2000
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R.E. JENSEN ET AL.
Fig. 1. Yeast mitochondrial tubules containing matrix-targeted
GFP. Plasmid pHS12 (Sesaki and Jensen, 1999), which encodes a
fusion protein consisting of the first 21 amino acids of cytochrome
oxidase subunit IV and the green fluorescent protein, was transformed into S. cerevisiae cells and examined by fluorescence microscopy. Differential interference contrast (DIC) and fluorescence
(COX4-GFP) and images of budding yeast cells are shown.
cells were examined by fluorescence microscopy. Although these screens proved successful, they were labor-intensive and tedious, allowing only the examination of a few thousand mutant colonies. Recently, a new
screening procedure has been developed that allows
the isolation of individual mutant cells with interesting
mitochondrial phenotypes from a large total population
of cells (Sesaki and Jensen, 1999). Specifically, yeast
cells were mutagenized and a suspension of cells was
examined using an inverted microscope. Individual
mutants with altered mitochondrial shape, number, or
location were isolated using a micropipette.
To visualize mitochondria in living cells, many genetic screens took advantage of fluorescent dyes specific for mitochondria, such as DASPMI (McConnell et
al., 1990), DiOC6 (Burgess et al., 1994; Hermann et al.,
1997), and DAPI (Meeusen et al., 1999). More recently,
the green fluorescent protein (GFP) and its derivatives
have been used to label mitochondria (Bleazard et al.,
1999; Hermann et al., 1998; Nunnari et al., 1997;
Sesaki and Jensen, 1999). For example, GFP has been
fused to the targeting signal of mitochondrial cytochrome oxidase subunit IV (COX4) and expressed in
yeast cells (Fig. 1; Sesaki and Jensen, 1999). Cells
containing COX4-GFP show uniform mitochondrial
fluorescence intensity, ideal for screening mutants or
for examining mitochondrial dynamics in wild-type
and mutant cells. Table 1 summarizes the yeast mutants identified to date using the genetic strategies
described above. Below we consider in more detail the
yeast mutants defective in mitochondrial fusion, division, segregation, and shape.
MITOCHONDRIAL FUSION AND DIVISION
Mitochondrial fusion and division have been directly
observed in many different eukaryotic cells (BereiterHahn, 1990; Bereiter-Hahn and Voth, 1994). For example, in cultured Xenopus endothelial cells, mitochondrial division was observed by time-lapse microscopy
(Bereiter-Hahn and Voth, 1994). The ability of mitochondria to fuse has also been demonstrated in cell-cell
fusion studies using differentially marked mitochondria (Hayashi et al., 1994) and in in vitro fusion assays
(Cortese, 1999). In addition to differentiated cells, mitochondrial fusion and fission are important during
development. Fusion leads to the formation of the complex mitochondrial networks seen in tissues, such as
muscle (Baldwin, 1984; Burleigh, 1974), liver (David,
1985) and heart (David et al., 1981; Smolich, 1995).
Previtellogenic oocytes of Xenopus contain large aggregates of mitochondrial tubules called the mitochondrial
cloud (Schnapp et al., 1997). At a later stage of oogenesis, the mitochondrial cloud divides and the resulting
fragments move to the vegetal pole (Heasman et al.,
1984). Division of mitochondria is clearly required for
inheritance in organisms, such as the ultramicroalga
Cyanidioschyzon merolae (Suzuki et al., 1994), that
contain only one mitochondrion per cell.
Membrane fusion and division in other parts of the
cell, such as those in the secretory pathway, have been
shown to use a well-studied collection of proteins that
includes coat proteins, SNAREs, NSF, and a family of
small GTPases (Mellman and Warren, 2000). None of
these components has yet been implicated in mitochondrial fusion, where very different machinery appears to
be utilized. In contrast, as discussed below, mitochondrial division requires a protein very similar to one
that is used in mammalian endocytosis.
Mitochondrial Fusion and Division in Yeast
The clearest examples of mitochondrial fusion in
yeast are seen following cell mating (Fig. 2), where the
mixing of mitochondrial contents, mtDNAs, and proteins, has been observed (Azpiroz and Butow, 1993;
Dujon et al., 1974; Nunnari et al., 1997; Okamoto et al.,
1998; Thomas and Wilkie, 1968; Wilkie and Thomas,
1973). Early studies showed that genetically marked
mtDNAs carried in each parent would recombine in
zygotes, indicating that the two mtDNA populations
had mixed (Azpiroz and Butow, 1993; Dujon et al.,
1974; Thomas and Wilkie, 1968; Wilkie and Thomas,
1973). Later experiments showed that mitochondrial
matrix proteins contained in one or both parental cells
would rapidly mix in the diploid zygote (Azpiroz and
Butow, 1993; Nunnari et al., 1997; Okamoto et al.,
1998). Interestingly, some mitochondrial components
do not freely diffuse following fusion. Mitochondrial
membrane proteins equilibrate at much slower rates
than matrix proteins (Okamoto et al., 1998). Similarly,
mtDNA does not appear to rapidly mix following mitochondrial fusion (Azpiroz and Butow, 1993; Nunnari et
al., 1997; Okamoto et al., 1998; Strausberg and Perlman, 1978; Zinn et al., 1987).
A striking example of mitochondrial fusion and fission is seen during yeast sporulation. When diploids
are induced to undergo meiosis and sporulation, a complicated series of morphogenetic changes occur in the
mitochondria (Fig. 3; Miyakawa et al., 1984; Smith et
al., 1995; Stevens, 1981). Upon starvation, mitochondrial tubules fragment into many (⬃ 30 –50) separate,
small organelles. Shortly after the onset of sporulation,
in premeitotic DNA synthesis, mitochondria fuse into a
single, intertwined tubule. This tubule then migrates
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YEAST MITOCHONDRIAL DYNAMICS
TABLE 1. Yeast proteins that appear to directly mediate mitochondrial dynamics
Protein
Mutant Phenotype
Cellular Location
Fzo1p
Fragmentation of mitochondria;
fusion defect
Mitochondrial outer
membrane
GTP-binding protein
Dnm1p
Inter-connected mitochondrial
tubules; division defect
Cytosol and
mitochondria
Dynamin-related GTPase
Mgm1p
Fragmentation of mitochondria;
defective transmission of
mitochondria
Defective transmission of
mitochondria; altered actin
distribution
Defective transmission of
mitochondria and nuclei
Defective transmission of
mitochondria to bud; altered
mitochondrial shape
Defective transmission of
mitochondria to bud; altered
mitochondrial shape
Defective transmission of
mitochondria to bud; altered
mitochondrial shape
Mitochondrial outer
membrane
Dynamin-related protein
Cytosol
Coiled-coil protein
Hermann et al., 1998
Rapaport et al., 1998
Bleazard et al., 1999
Sesaki and Jensen, 1999
Gammie et al., 1995
Otsuga et al., 1998
Sesaki and Jensen, 1999
Jones and Fangman, 1992
Guan et al., 1993
Shepard and Yaffe, 1999
Hermann et al., 1997
Cytosol
Intermediate filament-related
protein
Integral membrane protein
McConnell et al., 1990
McConnell and Yaffe, 1993
Burgess et. al., 1994
Mitochondrial outer
membrane
Integral membrane protein
Sogo and Yaffe, 1994
Mitochondrial outer
membrane
Integral membrane protein
Berger et. al., 1997
Mdm20p
Mdm1p
Mmm1p
Mdm10p
Mdm12p
Protein Properties
Mitochondrial outer
membrane
and divides, eventually encircling each of the nuclei of
the four spores after meiosis is complete.
Time-lapse video-microscopy of single yeast cells
showed that mitochondria frequently fuse and divide
during normal cell growth, with each occurring about
once every two minutes (Nunnari et al., 1997). Furthermore, fusion and fission appeared to be in rough equilibrium, so that the net number of mitochondria per
cell remained relatively constant. Fusion in yeast occurred between the tip of one mitochondrial tubule and
either the side or tip of another tubule. Division, on the
other hand, could happen anywhere along the length of
a mitochondrial tubule.
Mitochondrial Fusion Requires
the Fzo1 Protein
A major advance in our understanding of mitochondrial fusion came from work in Drosophila spermatogenesis, during which many small, spherical mitochondria aggregate and fuse into two helices that wrap
around each other at the base of flagella (Fuller, 1993).
In fuzzy onions mutants, sperm mitochondria fail to
fuse and instead occur as clusters of fragmented organelles (Hales and Fuller, 1997). fuzzy onions mutants were shown to be defective in a mitochondrial
transmembrane GTPase, required for mitochondrial
fusion in sperm. Consistent with this role, the fuzzy
onions protein appears on sperm mitochondria immediately before fusion and disappears quickly after fusion is complete. Another protein homologous to fuzzy
onions is present in the recently completed Drosophila
genome project (Adams et al., 2000). It remains to be
determined if this second GTPase mediates mitochondrial fusion in other tissues, instead of being specialized for spermatogenesis like fuzzy onions.
The yeast homologue of the Drosophila fuzzy onions,
FZO1, has been shown to play an essential role in
mitochondrial fusion (Bleazard et al., 1999; Hermann
et al., 1998; Rapaport et al., 1998; Sesaki and Jensen,
1999). Yeast cells lacking the Fzo1 protein contain
References
numerous mitochondrial fragments and rapidly lose
mitochondrial DNA (mtDNA). Using a mating assay
(see Fig. 2), fzo1 mutants were shown to be completely
defective in mitochondrial fusion (Bleazard et al., 1999;
Hermann et al., 1998; Sesaki and Jensen, 1999). The
Fzo1 protein is located in the mitochondrial outer
membrane with its GTPase domain facing the cytosol
(Hermann et al., 1998; Rapaport et al., 1998). Furthermore, the Fzo1 protein appears to be part of a multisubunit fusion complex on the mitochondrial surface
(Rapaport et al., 1998). Whether Fzo1p is critical for
mitochondrial fusion during sporulation is unclear.
However, it is interesting to note that FZO1 mRNA
expression is up-regulated at two different times during sporulation, which may correspond to periods of
increased fusion activity (Chu et al., 1998; Cerveny,
unpublished observations).
A number of questions remain about the role of the
Fzo1 protein in mitochondrial fusion. For example,
does Fzo1p directly catalyze the fusion reaction, or does
it play a regulatory role? During fusion, how is the
integrity of the mitochondrial outer and inner membranes maintained? If Fzo1p is evenly distributed over
the mitochondrial surface (Hermann et al., 1998), then
how is mitochondrial fusion restricted to the tip of one
or both organelles? What other components are required for fusion, and how are they regulated?
Dnm1 Protein Mediates Mitochondrial
Division in Yeast
Similar to fusion, division of yeast mitochondria requires a GTP-binding protein. This protein, called
Dnm1p, was first identified in yeast (Gammie et al.,
1995) as a homologue of dynamin, a component required for endocytosis in mammals and flies (Schmid et
al., 1998; van der Bliek, 1999). dnm1 mutants also
turned up in screens for yeast mutants defective in
mitochondrial morphology (Otsuga et al., 1998; Sesaki
and Jensen, 1999). Like dynamin, yeast Dnm1p contains a GTP-binding domain essential for its function
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R.E. JENSEN ET AL.
Fig. 2. Microscope-based assay for mitochondrial fusion during yeast
cell mating. Mitochondria of cells of one mating type are labeled with a
fluorescent marker, e.g., MATa cells carry matrix-targeted GFP. Mitochondria of cells of the opposite mating type are labeled with a different
fluorescent compound, e.g., MAT␣ cells are labeled with MitoTracker
Red. In the zygote, mitochondrial fusion will result in the mixing of the
two fluorescent markers. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
(Otsuga et al., 1998) and many dnm1 mutants carry
mutations in this region (Fig. 4). The best evidence that
Dnm1p is directly involved in mitochondrial division
comes from two recent studies (Bleazard et al., 1999;
Sesaki and Jensen, 1999). Sesaki and Jensen isolated
14 mutants in which the mitochondria lost their normal branched, tubular structure and instead formed a
single organelle consisting of interconnected tubules
(Fig. 5). These mutants all carried mutations in DNM1.
In addition, several fzo1 mutants were isolated, each
defective in mitochondrial fusion and containing numerous mitochondrial fragments (Fig. 5). Surprisingly,
the majority of dnm1 fzo1 double mutants contained
mitochondria with normal-looking tubules (Fig. 5). It
was proposed that Dnm1p plays a key role in mitochondrial division, and that mitochondrial number is controlled by a balance between division and fusion that
requires Dnm1p and Fzo1p, respectively. The dnm1
mutation also suppresses the mtDNA loss phenotype of
fzo1 cells, as dnm1 fzo1 mutants maintain mtDNA and
grow on nonfermentable carbon sources (Fig. 5; Bleazard et al., 1999).
Interestingly, while the number of mitochondria in a
given cell is controlled by the relative rates of fusion
and fission, mitochondrial shape is not. In dnm1 fzo1
cells, normal-looking mitochondrial tubules are seen.
Tubular shape must, therefore, arise from a mechanism independent of fusion and division, such as the
growth of mitochondria from the ends of pre-existing
organelles. Along similar lines, how are mitochondria
segregated to daughter cells in dnm1 (or dnm1 fzo1)
mutants? Sesaki and Jensen (1999) suggested that mitochondria in dnm1 mutants might be fragmented indirectly. For example, mitochondria may be pulled in
different directions by cytoskeletal interactions in the
mother and daughter cells, or cytokinesis itself may
pinch the organelles in half.
Arguing for a direct role in mitochondrial fission, at
least some of Dnm1 protein in the yeast cell appears to
be localized at sites of division. Bleazard et al. (1999)
showed by immuno-electron microscopy that a major
fraction of Dnm1p was clustered at sites of reduced
mitochondrial diameter, or at the ends of mitochondrial tubules. Similarly, a Dnm1p-GFP fusion protein
is found in punctate structures on the mitochondrial
surface (Fig. 6), many of which correlated with sites of
division (Sesaki and Jensen, 1999). Not all Dnm1p,
however, is located on the mitochondrial tubules (Otsuga et al., 1998). Using a Dnm1p-GFP fusion protein,
a pool of Dnm1p was found in the cytosol, located in
small, rapidly-moving dot-like structures, which could
not be captured in microscope images of living cells
(Aiken Hobbs, Sesaki and Cerveny, unpublished observations). Since Dnm1p has been reported to have a role
in endocytosis (Gammie et al., 1995), it is possible that
the cytoplasmic Dnm1p-containing spots are associ-
Fig. 3. Mitochondrial morphogenesis and
transmission during meiosis and sporulation.
When diploid (2N) cells enter stationary
phase, mitochondrial tubules fragment into
many, small organelles. During premeiotic S
phase (4N), mitochondria fuse into long,
branched tubules. After Meiosis I (not shown)
and Meiosis II (N), most of the tubules wrap
around the nuclei, and are then incorporated
into each of the four spores.
YEAST MITOCHONDRIAL DYNAMICS
Fig. 4. dnm1 mutations define three domains in Dnm1p. A: Sequence analysis of the DNM1 genes from different mutants. The DNA
mutations and the resulting amino acid substitutions are shown. B:
Distribution of mutations in Dnm1p. Amino acid substitutions shown
above the Dnm1p diagram lead to a recessive phenotype. Substitutions below the diagram cause a dominant defect. Mutations localized
to the GTPase domain, the conserved CGGARI motif, and the GTPase
effector domain (GED) are indicated.
ated with endocytic vesicles. However, it is also possible that the cytosolic structures represent Dnm1p-containing complexes prior to (or after) their association
with mitochondria. Supporting this view, Dnm1p-GFP
fusion proteins expressed from DNA from the dominant mutants, DNM1-110, DNM1-111, DNM1-112,
and DNM1-113, were found on mitochondria, but
lacked all cytosolic fluorescence (Fig. 6). These mutant
Dnm1 proteins appear to be “locked” on the mitochondrial surface.
Another dominant mutant, DNM1-109, was found to
completely disrupt the cellular distribution of Dnm1p.
A Dnm1-109p-GFP fusion shows a diffuse cytosolic fluorescence, and no punctate staining either in the cytosol or on the mitochondria (Fig. 6). The DNM1-109
mutation substitutes a glycine for an aspartic acid at
the position 385 of Dnm1p (Fig. 4) and resides immediately adjacent to a motif, CGGARI, conserved in
many different dynamin proteins (McNiven et al.,
2000). The Dnm1-109 protein, therefore, appears to be
defective in a region important for its association with
mitochondria.
577
Fig. 5. dnm1 fzo1 cells contain normal mitochondrial shape and
maintain mitochondrial DNA. A: Wild-type (BY4733), dnm1 (YHS19),
fzo1 (YHS21), and dnm1 fzo1 (YHS27) cells containing pHS12, which
expresses COX4-GFP, were examined by fluorescence microscopy as
described (Sesaki and Jensen, 1999). Bar ⫽ 2 ␮m. B: dnm1 fzo1 cells
contain mtDNA and are respiration competent. Wild-type, dnm1 (d⌬),
fzo1 (f⌬), and dnm1 fzo1 (d⌬f⌬) cells were struck onto YPD medium,
and YEPglycerol/ethanol medium (YPGE), and incubated at 30°C for
5 days.
Does Dnm1p Function as a Molecular
“Pinchase”?
Mammalian dynamin has been proposed to assemble
into spiral-like structures around plasma membrane
invaginations that then constrict “pinch” off endocytic
vesicles (Hinshaw and Schmid, 1995; Sweitzer and
Hinshaw, 1998; Takei et al., 1995, 1998). By analogy,
Dnm1p may directly catalyze the scission of mitochondria. Supporting this possibility, Dnm1p is located in
large mitochondrial-associated structures (see above).
In addition, many mutations in DNM1 are dominant
(Fig. 4), indicating that Dnm1p is part of a multisubunit complex. Further supporting a mechanical role
in division, time-lapse microscopy of C. elegans cells
showed that DRP-1, a Dnm1p-related protein, was located in clusters on the sides of mitochondrial tubules
where division eventually occurred (Labrousse et al.,
1999). Interestingly, cells disrupted for DRP-1 function
failed to divide the mitochondrial outer membrane, but
578
R.E. JENSEN ET AL.
Fig. 6. Localization of wild-type and mutant Dnm1p-GFP fusion
proteins. A: dnm1 strain YHS19 was transformed with plasmids
expressing Dnm1p-GFP from the following genes: wild-type (pHS20);
DNM1-109 (pHS21); DNM1-110 (pHS22); DNM1-111 (pHS23);
DNM1-112 (pHS24); DNM1-113 (pHS25); DNM1-114 (pHS26) empty
vector (pRS415). Cells were grown at 30°C in SGalactose medium to
an OD600 of 0.5– 0.8. Cells were labeled with 0.1 ␮M MitoTracker Red
CMXRos (Molecular Probes Inc., Eugene, OR) for 30 minutes, and
then examined by fluorescence microscopy. Merged images taken in
the red (MitoTracker) and green (GFP) channels are shown. Bar ⫽
2 ␮m. B: Dnm1p-GFP fusion proteins are intact in yeast cells. Total
cell protein from the above strains was subjected to SDS-PAGE.
Immune blots were decorated with antibodies to GFP and visualized
by chemiluminescence. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
scission of the inner membrane still occurred, raising
the possibility that division of the inner membrane
requires proteins distinct from DRP-1.
Although located at sites of fission, it is possible that
Dnm1p may not directly catalyze division, but instead
plays a regulatory role. Alterations of the GTPase effector
domain (GED) of dynamin, suggest that the assembled,
GTP-bound form of dynamin recruits and activates the
division machinery, and GTP hydrolysis results in the
disassembly of the inactive GDP-bound dynamin (Sever
et al., 1999). A possible candidate for recruitment by
dynamin is the endophilin protein (Schmidt et al., 1999).
Interestingly, yeast Dnm1p contains a GED domain, and
one dnm1 mutant carries a mutation near this region
(Fig. 4). Another potential function for Dnm1p comes
from studies with the mammalian Dnm1p homologue,
Dlp1 (Pitts et al., 1999). Dlp1 is located both on the
mitochondria and on small vesicles apparently derived
from the endoplasmic reticulum. Dlp1, and by analogy
Dnm1p, may shuttle materials such as lipids between the
ER and the mitochondria, thereby playing only an indirect role in mitochondrial fission.
YEAST MITOCHONDRIAL DYNAMICS
Although mitochondria are thought to have arisen
from bacterial endosymbionts in primitive eukaryotic
cells (Gray et al., 1999; Margulis, 1975), it appears that
in many cases the eukaryotic cell has discarded the
bacterial fission machinery. Virtually all bacterial cells
use a protein called FtsZ for cytokinesis, which forms a
contractile ring at the septation furrow (Erickson,
2000). However, there is no FtsZ in the completed
genomes of S. cerevisiae and Drosophila melanogaster.
In contrast to mitochondria, chloroplasts do appear to
use FtsZ for division (Osteryoung and Pyke, 1998;
Strepp et al., 1998). In fact they use two different FtsZ
proteins, one on the surface of the organelle, and one on
the inside of the inner membrane. Recently, the alga
M. splendens has been found to contain a protein related to FtsZ containing a mitochondrial targeting signal (Beech et al., 2000). Furthermore, two parasitic
bacteria were found to lack FtsZ, but instead may use
the host cell’s dynamin for their division (Boleti et al.,
1999). Although Dnm1p and FtsZ are both GTP-binding proteins, they do not seem to be significantly homologous to each other. Nonetheless, it is possible that
both proteins carry out the same function, and eukaryotic cells use either a Dnm1p or FtsZ-based machines
for division of their mitochondria.
Besides Dnm1p, yeast cells contain another mitochondrial-associated, dynamin-like protein, Mgm1p
(Guan et al., 1993; Jones and Fangman, 1992; Shepard
and Yaffe, 1999). Based on its homology, it is tempting
to speculate that Mgm1p plays a role in mitochondrial
division. However, it is likely that Mgm1p is involved
in some other mitochondrial process instead of fission.
For example, mgm1 mutants contain fragmented mitochondria, instead of the single, interconnected network of tubules seen in dnm1 mutants. In addition,
mgm1 mutants are defective in mitochondrial inheritance, whereas dnm1 cells show no segregation defect.
The Mgm1 protein has been reported to reside in the
mitochondrial outer membrane (Shepard and Yaffe,
1999). However, a homologous protein in S. pombe
appears to be located in the mitochondrial inner membrane (Pelloquin et al., 1998). Consequently, the location and function of Mgm1p await further analyses.
SEGREGATION OF MITOCHONDRIA DURING
CELL DIVISION
Mitochondria are essential organelles and consequently their segregation to daughter cells following
cytokinesis is a critical event. Many observations indicate that mitochondria are attached to the cytoskeleton, and it is likely that this connection is important for
organelle inheritance. In different cell types, mitochondria have been shown to colocalize with all three cytoskeletal elements: actin filaments (Drubin et al.,
1993; Pardo et al., 1983), microtubules (Baumann and
Murphy, 1995; Couchman and Rees, 1982; Heggeness
et al., 1978; Schnapp and Reese, 1982; Summerhayes
et al., 1983), and intermediate filaments (Collier et al.,
1993; Leterrier et al., 1994; Morse-Larsen et al., 1982;
Summerhayes et al., 1983). In many cells, microtubules have been linked to mitochondrial transmission.
For example, microtubule inhibitors block mitochondrial movement in neuronal cells (Morris and Hollenbeck, 1993), and mutant alleles of S. pombe tubulin
cause aberrant mitochondrial distribution (Yaffe et al.,
579
1996). In both mice and flies, members of the kinesin
family are located on mitochondria and are required for
in vitro motility or normal mitochondrial distribution
in vivo (Nangaku et al., 1994; Pereira et al., 1997). In
some cases, mitochondrial association or mobility
seems to require an interplay between microtubules
and actin (Couchman and Rees, 1982; Morris and Hollenbeck, 1995), or between microtubules and intermediate filaments (Summerhayes et al., 1983), but the
nature of these interactions is unknown.
Yeast Mitochondria and the Cytoskeleton
In S. cerevesiae, transmission of mitochondria appears to require the actin cytoskeleton, but not microtubules. Some actin alleles alter mitochondrial shape
and distribution, and a fraction of mitochondria colocalize with actin cables (Drubin et al., 1993). Depolymerization of microtubules, on the other hand, does not
affect mitochondrial segregation (Burgess et al., 1994;
Smith et al., 1995). Isolated yeast mitochondria were
found to bind actin filaments and also showed actinbased motility (Lazzarino et al., 1994; Simon et al.,
1995). Further supporting a role for actin in transmission, mdm20 mutants are defective in both mitochondrial segregation to daughter cells and in normal actin
organization, and a yeast tropomyosin-like protein can
suppress the mdm20 defect (Hermann et al., 1997).
The link between actin and mitochondrial movement,
however, is not completely clear. Some yeast mutants
that are deficient in actin cables still efficiently transmit mitochondria to daughter cells (Drubin et al., 1993;
Liu and Bretscher, 1989). Moreover, yeast cells lacking
any of the five yeast myosins, likely motors for actinbased movement, have no effect on mitochondrial segregation (Goodson et al., 1996; Simon et al., 1995).
In addition to its apparent role in mitochondrial segregation, the actin cytoskeleton may also be important
for mitochondrial division and fusion. In particular,
yeast cells treated with latrunculin A, which completely disrupts the actin cytoskeleton (Ayscough et al.,
1997), quickly fragment their mitochondria (Boldogh et
al., 1998). Instead of normal tubules, cells incubated
with latrunculin A contain numerous, irregular-shaped
organelles (Fig. 7). Surprisingly, this fragmentation is
dependent upon Dnm1p (Fig. 7). dnm1 mutants contain a single organelle consisting of interconnected tubules, which is sometimes partially collapsed to one
side of the cell (Otsuga et al., 1998; Sesaki and Jensen,
1999). The tubular network in dnm1 mutants treated
with latrunculin A is more open and dispersed than in
untreated cells, supporting the idea that mitochondria
are normally bound to actin. It is clear, however, that
the mitochondrial network in dnm1 cells is not fragmented, but instead remains interconnected. These results suggest that in the absence of normal actin organization, mitochondrial division is activated in a
Dnm1p-dependent manner. Alternatively, mitochondrial fusion, but not fission, may require attachments
to actin. In either case, further studies are needed to
understand the interplay between the cytoskeleton and
mitochondrial division and fusion.
Mitochondrial transmission in yeast also appears to
require a protein related to mammalian intermediate
filament subunits. Mdm1p is an essential protein required for segregation of both nuclei and mitochondria,
580
R.E. JENSEN ET AL.
Fig. 7. Latrunculin A-induced fragmentation of mitochondria requires the Dnm1 protein. Wild-type and dnm1⌬ cells expressing
COX4-GFP (Sesaki and Jensen, 1999) were either mock treated, or
incubated with 250 ␮M latrunculin A (Molecular Probes Inc., Eugene,
OR) for at least 30 minutes, which was sufficient to completely disrupt
the actin cytoskeleton (not shown). Fluorescence images of representative cells are shown.
and is most homologous to mammalian vimentin (McConnell et al., 1990; McConnell and Yaffe, 1993). The
function of the Mdm1 protein, however, is not clear.
Mdm1p in yeast cells is located in small, dot-like structures throughout the cytosol (McConnell and Yaffe,
1992), while the Mdm1 protein, like many intermediate
filament proteins, forms 10-nm filaments in vitro (McConnell and Yaffe, 1993). Furthermore, since motor
proteins for intermediate filaments have not been identified, Mdm1p may not directly mediate mitochondrial
motility, but instead may play a more structural role in
inheritance. For example, Mdm1p may anchor mitochondria or the cytoskeleton to specific cellular locations.
mtDNA Segregation
Although most mitochondrial proteins are encoded in
the nucleus, the mitochondrial genome (mtDNA) in
many organisms, including yeast and mammals, encodes several proteins essential for respiration, as well
as the rRNAs and tRNAs required for their synthesis
(Attardi and Schatz, 1988; Wallace, 1999). While it is
critical that mtDNA is faithfully transmitted to daughter cells, the segregation mechanism is not yet known.
Yeast cells contain 25–50 copies of mtDNA, packaged
into about 10 –30 DNA/protein complexes called nucleoids (Fig. 8; Azpiroz and Butow, 1995; Williamson and
Fennell, 1979). As described above, in contrast to matrix proteins, mtDNA does not quickly equilibrate following mitochondrial fusion after mating (Azpiroz and
Butow, 1993; Birky et al., 1978; Nunnari et al., 1997;
Okamoto et al., 1998; Strausberg and Perlman, 1978;
Zinn et al., 1987). These studies indicate that mixing of
marked mtDNAs is restricted to the buds adjacent to
Fig. 8. mtDNA nucleoids in budding yeast cells. Wild-type yeast
cells from a logarithmically growing culture were grown in YEPglycerol/ethanol medium, stained with 0.5 ␮g/ml DAPI and examined by
fluorescence microscopy. Nucleoids are seen as punctate structures
within the mitochondrial tubules.
the zygote neck, and buds from one or the other end of
the zygote most often contain only one type of mtDNA.
It is likely that mtDNA is attached to the mitochondrial inner membrane and that this attachment is important for DNA segregation. Several proteins likely to
play a role in mtDNA attachment, nucleoid formation,
or segregation have been identified (Chen et al., 1993;
Diffley and Stillman, 1991; 1992; Meeusen et al., 1999;
Newman et al., 1996; Piskur, 1997; Xiao and Samson,
1992; Zweifel and Fangman, 1991), but their function
in mtDNA inheritance has yet to be determined.
Mitochondrial Proteins Required for Both
Shape and Segregation
Three yeast proteins, Mmm1p, Mdm10p and
Mdm12p, have been identified that are important for
normal mitochondrial shape, mtDNA maintenance,
and the segregation of mitochondria to daughter cells.
In mmm1 (Burgess et al., 1994), mdm10 (Sogo and
Yaffe, 1994), and mdm12 (Berger et al., 1997) mutants,
the elongated, branched structure of mitochondria is no
longer seen. Mitochondria instead appear as a few
large, spherical organelles (Fig. 9). Loss of Mmm1p,
Mdm10p, or Mdm12p function also causes the rapid
loss of mtDNA. Furthermore, the altered mitochondria
in mmm1, mdm10, and mdm12 mutants are not efficiently transmitted to daughter buds following cytokinesis. Recently, the Mmm1 protein has been implicated
in playing a role in the retention of a subset of mitochondria at the base of the mother cell during cytokinesis (Yang et al., 1999).
The Mmm1, Mdm10, and Mdm12 proteins all reside
in the mitochondrial outer membrane (Berger et al.,
1997; Burgess et al., 1994; Sogo and Yaffe, 1994), and
genetic and biochemical studies suggest that all three
proteins may be subunits of an oligomeric complex. For
example, double mutants between mmm1, mdm10, or
mdm12 show the same mitochondrial phenotype
581
YEAST MITOCHONDRIAL DYNAMICS
published observations), in which mitochondria appear
to interact with microtubules instead of actin (Yaffe et
al., 1996). Furthermore, defects in a mitochondrialactin association do not explain why mtDNA is rapidly
lost in mmm1, mdm10, and mdm12 mutants. The functions of Mmm1p, Mdm10p, and Mdm12p clearly await
further analyses.
Fig. 9. Mitochondrial shape in wild-type, mmm1 and mdm10 cells.
Wild-type and mmm1::URA3 cells (Burgess et al., 1994) contain matrix-targeted GFP from plasmid pOK29 (Sesaki and Jensen, 1999). A
recently-isolated mdm10 mutant, contains chromosomal COX4-GFP
(Sesaki and Jensen, 1999). Fluorescence images of representative
cells are shown.
FUTURE CHALLENGES
Although a handful of proteins that mediate mitochondrial dynamics have been identified, it is obvious
that we have only scratched the surface. Continued
isolation and analysis of yeast mutants, as well as
further studies in other organisms, are needed to expand the repertoire of proteins required for mitochondrial fusion, division, segregation, and shape. Understanding these processes also requires additional assays with which to examine the function of these
proteins. To determine the mechanisms of mitochondrial fusion and division, it is critical that we reconstitute these reactions in vitro. In addition to their functions, it is also important to understand how the activities of these different proteins are regulated by the
cell. To keep mitochondrial fusion and division in balance, there must be exquisite control of both processes.
Since mitochondria are essential organelles, the cell
must have a means of ensuring and monitoring transmission of both the organelle and its enclosed mtDNA.
Finally, since defects in mitochondrial shape and distribution (Yaffe, 1999), and in the maintenance of
mtDNA (Fliss et al., 2000; Marin-Garcia and Goldenthal, 2000; Polyak et al., 1998), are associated with a
number of human diseases, including neuromuscular
disorders, cardiomyopathies, liver disease, and cancer,
our understanding of mitochondrial dynamics will
have a wide-ranging impact.
(Berger et al., 1997). Each are lethal in combination
with phb1 or phb2 mutations (Berger and Yaffe, 1998),
and mmm1, mdm10, and mdm12 can each be suppressed by an alteration in the Sot1 protein (Berger et
al., 1997). Blue native electrophoresis of detergentsolubilized mitochondria indicates that the 48 kDa
Mmm1 protein migrates at ⬎200 kDa (A. Aiken Hobbs,
unpublished observations). It has been suggested that
Mmm1p, Mdm10p, and Mdm12p maintain mitochondria in an elongated conformation by mediating an
interaction between the organelle and the cytoskeleton.
Supporting this possibility, mitochondria isolated from
mmm1 and mdm10 mutants are defective in their
binding to actin filaments and also in actin-based motility assays (Boldogh et al., 1998).
The function of Mmm1p, Mdm10p, and Mdm12p cannot simply be to connect mitochondria to actin. Disruption of the actin cytoskeleton in yeast cells with latrunculin A causes alterations in mitochondrial shape due
to their fragmentation, but does not produce the large,
spherical organelles seen in mmm1, mdm10, or mdm12
mutants (compare Figs. 7 and 8; Boldogh et al., 1998;
A. Aiken Hobbs, unpublished observations). In addition, homologues of Mmm1p, Mdm10p, and Mdm12p
are found in S. pombe (Berger et al., 1997; Berger and
Yaffe, 1996; Jamet-Vierny et al., 1997: R. Jensen, un-
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