Department of Biology, University of California, San Diego, La Jolla, California 92093
Nuclear and mitochondrial transmission to daughter buds of Saccharomyces cerevisiae depends on Mdm1p, an intermediate filament-like protein localized to numerous punctate structures distributed throughout the yeast cell cytoplasm. These structures disappear and organelle inheritance is disrupted when mdm1 mutant cells are incubated at the restrictive temperature. To characterize further the function of Mdm1p, new mutant mdm1 alleles that confer temperature-sensitive growth and defects in organelle inheritance but produce stable Mdm1p structures were isolated. Microscopic analysis of the new mdm1 mutants revealed three phenotypic classes: Class I mutants showed defects in both mitochondrial and nuclear transmission; Class II alleles displayed defective mitochondrial inheritance but had no effect on nuclear movement; and Class III mutants showed aberrant nuclear inheritance but normal mitochondrial distribution. Class I and II mutants also exhibited altered mitochondrial morphology, possessing primarily small, round mitochondria instead of the extended tubular structures found in wild-type cells. Mutant mdm1 alleles affecting nuclear transmission were of two types: Class Ia and IIIa mutants were deficient for nuclear movement into daughter buds, while Class Ib and IIIb mutants displayed a complete transfer of all nuclear DNA into buds. The mutations defining all three allelic classes mapped to two distinct domains within the Mdm1p protein. Genetic crosses of yeast strains containing different mdm1 alleles revealed complex genetic interactions including intragenic suppression, synthetic phenotypes, and intragenic complementation. These results support a model of Mdm1p function in which a network comprised of multimeric assemblies of the protein mediates two distinct cellular processes.
CYTOPLASMIC organelles are propagated by growth
and division of preexisting organelles (Palade,
1983 Nuclear and mitochondrial inheritance in the yeast Saccharomyces cerevisiae depends on Mdm1p, an intermediate
filament-like protein that defines a series of punctate
structures distributed throughout the yeast cytoplasm
(McConnell and Yaffe, 1992 To explore Mdm1p function further, we have generated
new mdm1 mutant alleles that cause defects in organelle
inheritance but yield stable Mdm1p punctate structures
even during incubation of cells at the nonpermissive temperature. These novel alleles have facilitated a genetic dissection of Mdm1p functions in nuclear and mitochondrial
inheritance.
Yeast Strains and Genetic Methods
S. cerevisiae strains used in this study are listed in Table I. Strain MYY404
is a diploid in which one copy of MDM1 is replaced by the LEU2 gene and
was derived from MYY298 as described (McConnell and Yaffe, 1992 Table I.
Yeast Strains
; Yaffe, 1991
; Warren and Wickner, 1996
), so
an essential feature of cell proliferation is the inheritance
of organelles by daughter cells. Organelle inheritance is
thought to depend on functions of the cytoskeleton. Such a
role for cytoskeletal components has been suggested by microscopic studies that revealed colocalization of organelles with microtubules (Heggeness et al., 1978
; Ball
and Singer, 1982
; Couchman and Rees, 1982
), intermediate filaments (David-Ferreira and David-Ferreira, 1980
;
Mose-Larsen et al., 1982
; Chen, 1988
), or actin microfilaments (Wang and Goldman, 1978
; Kachar and Reese, 1988
)
in various types of cells. In addition, studies in vitro have
indicated possible functions of microtubule-based motor
proteins (Vale, 1987
) or unconventional myosins (Adams
and Pollard, 1986
; Allan, 1995
) in facilitating organelle movement. However, many details of the activity and roles of
particular cytoskeletal components in mediating organelle
movement and distribution in living cells remain obscure.
, 1993
). The punctate Mdm1p
structures disappear at 37°C in cells harboring the temperature-sensitive mdm1-1 mutation (McConnell and Yaffe, 1992
), and this disappearance coincides with a failure to
transmit mitochondria from the mother portion of the cell
into the growing bud. Additionally, the mdm1-1 lesion
causes a disorientation of the mitotic spindle such that nuclear division occurs entirely within the mother portion of
the cell (McConnell et al., 1990
). These defects indicate
that the Mdm1p network has a central function in facilitating organelle inheritance; however, the mechanism of Mdm1p
function is unknown (Berger and Yaffe, 1996
).
Materials and Methods
).
Strain MYY404-1b was created by transforming MYY404 with plasmid
YCp50-MDM1 (McConnell and Yaffe, 1992
), followed by sporulation and
recovery of a spore that was MATa, Leu+, Ura+. Strains MYY700
through MYY721 were generated in the MYY290 (Smith and Yaffe, 1991
)
background by replacing the wild-type, chromosomal copy of MDM1 with
different mutant alleles, as described below. Strains MYY725 through
MYY746 were derived as temperature-sensitive, MAT
spores from
crosses of strain MYY291 with strains MYY700 through MYY721. Yeast
culture media were prepared, and standard genetic manipulations were
carried out as previously described (Rose et al., 1990
). Plasmid DNA was
prepared in Escherichia coli strain DH5
.
Construction of New mdm1 Alleles
Plasmid pMDM1 was constructed by subcloning the 2.1-kb SalI-EcoRV
fragment containing the MDM1 gene from plasmid YCp50-MDM1 into
the SalI and EcoRV sites of plasmid pRS423 (Sikorski and Hieter, 1989).
Plasmid pMDM1 was mutagenized in vitro with hydroxylamine as described by Sikorski and Boeke (1991)
.
Mutagenized, plasmid-borne copies of MDM1 that conferred temperature-sensitive growth on cells that harbored no other copy of MDM1 were
identified by a "plasmid shuffling" protocol similar to that described by
Sikorski and Boeke (1991). Briefly, MYY404-1b cells were transformed
with the pool of mutagenized pMDM1 DNA. Loss of the plasmid YCp50-MDM1 containing the URA3 gene and the wild-type copy of MDM1 was
selected by culturing on medium containing 5-fluoro-orotic acid (FOA).1
Cells resistant to FOA were tested for growth at 37°C, and 75 independently derived, temperature-sensitive isolates were identified. Plasmids
encoding various MDM1 alleles were recovered and amplified in bacterial cells.
New mutant versions of MDM1 were integrated at the chromosomal
MDM1 locus of haploid cells by a two-step gene replacement strategy.
DNA sequences encoding mdm1 alleles were excised from plasmid
pMDM1 by digestion with SphI and NheI and subcloning into the SphI
and NheI sites in the yeast integrating vector YIp5 (Struhl et al., 1979).
The YIp5 constructs were linearized at the unique BglII site in the MDM1
gene; linearized plasmids were transformed into MYY290; and Ura+
transformants were isolated. Correct integration of YIp5 at the MDM1 locus was confirmed by PCR analysis and by Southern blotting.
Excision of YIp5 and wild-type MDM1 sequences was selected by plating cells on medium containing FOA. The resulting Ura isolates were
tested for growth on YPD medium at 37°C, and temperature-sensitive strains were identified as candidate mutants. Temperature-sensitive growth of the resulting strains was shown to be caused by mutations in
MDM1 by the following analyses. Recessive mutants were complemented by transformation with unmutagenized MDM1 (pMDM1). Dominant mutants were mated to MYY403 (mdm1-1); the resulting diploids were
sporulated; and all meiotic progeny were shown to be temperature sensitive for growth. Further, a 2:2 ratio of segregation of temperature sensitivity was observed in meiotic progeny derived from a cross of mdm1 mutants to the wild-type strain MYY291. The presence of a single copy of
MDM1 (as well as the absence of YIp5 sequences) was verified by PCR
and Southern blot analyses of genomic DNA isolated from each mutant.
New mutant alleles of MDM1 were characterized by DNA sequence
analysis as follows. Genomic DNA was isolated from mutant cells harboring each allele. Asymmetric PCR (McCabe, 1990) was performed using
different ratios of the primers 5
-CTCAGCTCTTTGGTTCAG-3
and 5
-GGCTGAAGAAGTGTACC-3
flanking the MDM1 ORF to create predominantly single-stranded products corresponding to either the coding
or noncoding strand of MDM1. Nucleotide sequences of the PCR products were determined by dideoxy sequencing. All mutations were verified by sequencing both coding and noncoding strands derived from independent PCR reactions.
Phenotypic Analysis
Fluorescence and immunofluorescence microscopy to visualize mitochondria, microtubules, nuclei, and Mdm1p structures was performed as previously described (McConnell et al., 1990; McConnell and Yaffe, 1992
). To
visualize bud scars in conjunction with indirect immunofluorescence, cells
were incubated in a solution of 10 µg/ml Calcofluor (Sigma Chemical Co.,
St. Louis, MO) for 10 min before the final wash step. In some experiments, cell populations were synchronized in the cell cycle by treatment
with the yeast mating pheromone
-factor as previously described (McConnell et al., 1990
). Electron microscopy was performed as described previously (Sogo and Yaffe, 1994
).
Isolation of New mdm1 Alleles
Yeast strains harboring new mdm1 mutant alleles were generated by in vitro mutagenesis of the cloned MDM1 gene followed by replacement of the chromosomal, wild-type MDM1 gene with a mutated copy. This was accomplished in two steps (described in detail in Materials and Methods). First, mutagenized plasmids containing the MDM1 gene were screened for their ability to confer temperature-sensitive growth on cells that contained no other copy of MDM1. Second, candidate mdm1 alleles were integrated at the chromosomal MDM1 locus using a two-step allele replacement strategy. Correct integration and gene replacement were verified by PCR analysis, Southern blotting, and characterization of meiotic progeny derived from crosses of the new mutant strains to the mdm1-1 mutant. 75 independently isolated plasmids conferring temperature-sensitive growth were identified. Of these, mdm1 sequences from 13 were integrated at the MDM1 locus, and 10 mutant alleles that appeared to represent the range of phenotypic variations observed (as described below) were characterized in detail.
The dominant or recessive character of the new mdm1 alleles was determined by examining the growth of heterozygous (MDM1/mdm1) diploids obtained by crossing the mutant strains to the wild-type parent of opposite mating type. Six of the heterozygous diploid strains failed to grow at 37°C and therefore harbored dominant mutations, while the remaining four diploid strains grew at 37°C and therefore contained recessive alleles (Table II). The growth defects conferred by the recessive haploid alleles could be complemented by transformation of the strains with a centromere-based vector containing the wild-type MDM1 gene.
Table II. New mdm1 Alleles |
Uncoupling of Nuclear and Mitochondrial Inheritance Defects
To characterize the effects of the new mutations on organelle inheritance, cells were analyzed by fluorescence
and indirect immunofluorescence microscopy (Fig. 1). An
initial examination of cells treated with the mitochondria-specific, vital dye, 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI), revealed that only a subset of
the new alleles caused defects in mitochondrial distribution. Further analysis of both nuclei and mitochondria by
indirect immunofluorescence microscopy permitted the
assignment of mutant alleles to three phenotypic classes
(Table II). Whereas wild-type cells incubated at 37°C displayed normal organelle distribution and stable Mdm1p structures (Fig. 1 A), Class I mutants displayed defects in
both mitochondrial and nuclear transmission to buds (Fig.
1 B), similar to the original mdm1-1 allele. Class II alleles
appeared to affect only mitochondrial inheritance, with
normal nuclear behavior (Fig. 1 C). Finally, Class III mutants displayed aberrant nuclear transmission but normal
mitochondrial distribution (Fig. 1 D). Both dominant and
recessive Class I and Class III alleles were identified, but both Class II alleles obtained were dominant. All of the
mdm1 mutant cells possessed stable Mdm1p-punctate
structures (Fig. 1, B-D), in contrast to the Mdm1p structures observed in the original mdm1-1 strain, which disappeared after incubation at 37°C (McConnell and Yaffe,
1992). Additionally, immunoblot analysis of cellular extracts revealed no difference in Mdm1p levels between
wild-type and mutant cells grown at the permissive or nonpermissive temperature (data not shown), indicating that
the new mutant phenotypes were not caused by unstable
forms of Mdm1p. In some cells there appeared to be a concentration of Mdm1p staining at or around the nucleus; this staining was observed in both wild-type and mutant
cells and did not appear to correspond to a particular
phase of the cell cycle. Like the original mdm1-1 mutant,
none of the new mutant alleles appeared to alter actin distribution (to cortical patches and cables) at either permissive or nonpermissive temperature (data not shown).
Class I and Class II Mutants Display Aberrant Mitochondrial Morphology
In addition to a defect in mitochondrial distribution, microscopic analysis of Class I and Class II mutants revealed
altered mitochondrial morphology. Instead of the wild-type, tubular mitochondrial profiles (Fig. 1 A), mitochondria
in these mdm1 mutants appeared to be round structures of
fairly uniform size (Fig. 1, B and C). This morphology was
confirmed for a Class II mutant, mdm1-252 (MYY721), by
electron microscopy (Fig. 2). The round mitochondria frequently appeared clumped together in the mother portion
of a dividing cell and were often localized to the end opposite the emerging bud. Wild-type mitochondrial morphology was observed in Class II mutant strains cultured at
permissive temperature (data not shown). After the shift
of a Class II mutant culture to 37°C, cells with empty buds
began appearing in the population after 1 h; cells with
small, round mitochondria were apparent after 2 h; and
the majority of cells displayed both empty buds and small,
round mitochondria by 4 h (data not shown).
Class I and Class III Mutant Alleles Produce Two Distinct Nuclear Transmission Defects
Characterization of the different Class I and Class III alleles
revealed two types of defect in nuclear transmission. Some alleles (Class Ia and Class IIIa) were defective for the
movement of nuclei into daughter buds (Fig. 1, B and D).
Additionally, nuclear division occurred in a fraction of
these cells, yielding two nuclei in the mother portion of the
cell. These phenotypes are similar to those described for
the mdm1-1 mutant (McConnell et al., 1990).
A distinct, novel nuclear phenotype was displayed by several other alleles (Class Ib and Class IIIb). In these strains, a significant fraction of the cells incubated at the nonpermissive temperature exhibited all of the nuclear DNA localized to the bud (Fig. 3, A and B). Double-label experiments in which cells were stained with both DAPI (to reveal DNA) and Calcofluor (to label bud scars on the mother portion of the cells) confirmed that daughter buds were, in fact, receiving all of the nuclear DNA in these cells (Fig. 3 C). Visualization of microtubules in Class Ib and Class IIIb cells by indirect immunofluorescence microscopy revealed that many of the nuclei in the buds possessed unelongated mitotic spindles (Fig. 3 C), indicating aberrant positioning of the premitotic spindle apparatus.
To characterize the Class IIIb phenotype in greater detail, mutant cells were synchronized in the G1 (unbudded)
stage of the cell cycle, and nuclear distribution was analyzed 2 and 4 h after release from the cell cycle block at the
non permissive temperature. After both 2 and 4 h, 70-82%
of budded cells harboring either of two different Class IIIa
alleles (mdm1-200 or mdm1-217) possessed nuclei confined to the mother portion of the cell (Fig. 4). In contrast,
a substantial fraction (24-34%) of budded cells harboring either of two Class IIIb alleles (mdm1-227 or mdm1-228)
displayed nuclear DNA entirely transferred to the bud after 2 h (Fig. 4), and this fraction increased further (to 31-
38%) by 4 h. Correspondingly, the fraction of cells possessing properly divided nuclei distributed between the
mother and bud was significantly reduced in both Class IIIa and Class IIIb cultures compared to wild-type cells.
Certain Combinations of mdm1 Alleles Exhibit Intragenic Complementation and Suppression
During initial genetic analysis, diploids generated by
crosses of all the new mdm1 alleles to the original mdm1-1
mutant strain were found to grow at 37°C, suggesting intragenic complementation (data not shown). However,
crosses of new alleles to the wild-type strain from which
the mdm1-1 mutant was derived (A364a) (McConnell et
al., 1990) revealed that alleles found to be dominant in the MYY290 background behaved as recessive mutations in
the hybrid strain. Subsequent attempts to reconstruct the
mdm1-1 mutation in the genetic background of the new alleles (strain MYY290) were unsuccessful, and this failure
was consistent with the mdm1-1 mutation being lethal in
strain MYY290 (data not shown). Because of these strain
background variations, subsequent analyses were limited
to the new alleles in an otherwise isogenic MYY290 background.
To examine in greater detail the interactions between different mdm1 mutant alleles, all possible diploid combinations of the 10 new alleles were generated, and phenotypes of the resulting diploids were analyzed. Cells were examined for growth at 37°C and by fluorescence microscopy for mitochondrial and nuclear distribution. The consequent mutant phenotypes revealed by this analysis indicated a complex pattern of interactions (Fig. 5 A). First, certain combinations of dominant alleles yielded an apparently additive (and expected) phenotype. For example, the combination of either dominant Class II mutation with the dominant Class IIIb mutation, mdm1-227, produced diploids displaying a Class Ib phenotype. Second, some combinations of alleles yielded unexpected synthetic phenotypes. For example, the diploid obtained by crossing the recessive Class Ia mutant, mdm1-199, with the dominant Class II mutant, mdm1-202, displayed a Class Ib phenotype. Third, certain crosses led to intragenic suppression in which a phenotype caused by a dominant mutation was not observed in the diploid. An example of such intragenic suppression was the cross of the dominant Class II mutant, mdm1-202, with the dominant Class IIIa mutant, mdm1-200, resulting in a Class IIIb diploid that displayed no defect in mitochondrial distribution (suppression of the mitochondrial defect). A final type of interaction was intragenic complementation, in which the combination of two different alleles eliminated mutant phenotypes observed in one or both of the parental strains. An example of such complementation emerged from the mating of recessive Class IIIa mutant, mdm1-217, with recessive Class IIIb mutant, mdm1-228, to yield a diploid strain with wild-type growth and normal organelle distribution (Fig. 5 B).
Sequence Analysis
Mutations corresponding to the new mdm1 alleles were determined by nucleotide sequence analysis and are listed in Table III. Each mdm1 allele consists of a single mutation, with the exception of mdm1-251, a Class Ia allele containing R187Q and E378K. The R187Q mutation was identified also in the Class II allele, mdm1-252, and is therefore likely to be responsible for the mitochondrial inheritance defect in mdm1-251. Consistent with this finding, diploids expressing both mdm1-251 and mdm1-252 display a Class II phenotype (Fig. 5 A). These observations suggest that the E378K mutation causes a recessive, type "a" nuclear segregation defect (which is suppressed in the diploid).
Table III. Sequence Analysis of New mdm1 Alleles |
The mutations found in the various mdm1 alleles
mapped to two discrete clusters within the MDM1 open
reading frame (Fig. 6). These lesions did not appear to
cluster with respect to phenotype, as mutations conferring
all three phenotypic classes were found in both regions.
Previously, Mdm1p was identified as a component of a
novel system of cytoplasmic structures essential for nuclear and mitochondrial inheritance in yeast (McConnell
et al., 1990; McConnell and Yaffe, 1992
, 1993
). These earlier investigations revealed that incubation of the mdm1-1
mutant at the nonpermissive temperature led to defects in
nuclear and mitochondrial transmission to daughter buds
and to the disappearance of cytoplasmic, punctate structures defined by Mdm1p. We have now separated the assembly and stability of these Mdm1p structures from their
function in organelle inheritance by the isolation and analysis of new mdm1 alleles. Furthermore, these new alleles
define three phenotypic classes (summarized in Fig. 7). At
restrictive temperature, cells harboring these new mutant
alleles possess stable Mdm1p punctate structures yet display aberrant organelle inheritance. Although currently
available techniques do not allow the identification of
punctate structures that might be incorrectly assembled,
the successful transmission of nuclei in Class II mutants
and of mitochondria in Class III mutants implies that some
property other than network assembly (e.g., specific binding of an interacting protein) is affected by the new mutations.
The specific mutations that give rise to the new mdm1 alleles alter residues in two distinct regions within the Mdm1p protein (Fig. 6). Each of these regions contains mutations yielding each phenotypic class, so our analysis does not indicate that distinct structural domains of Mdm1p are specialized for function in nuclear or mitochondrial inheritance. Rather, various mdm1 mutations may differentially affect the binding affinity of proteins that link mitochondria or nuclei to Mdm1p structures. Thus, the new mdm1 lesions appear to define two distinct binding sites for the interaction of Mdm1p with other cellular structures.
Class I and Class III mdm1 mutations cause aberrant
positioning of the mitotic spindle leading to defective nuclear transmission. Different alleles conferring this phenotype display two strikingly different consequences: type
"a" alleles (similar to the original mdm1-1 allele) show nuclear DNA entirely confined to the mother portion of the
cell; while type "b" alleles show the complete transfer of
all nuclear DNA into daughter buds. In a fraction of cells
of both types, nuclear division occurs to yield two nuclei confined to either the mother or bud, respectively. Similar
nuclear phenotypes have been observed for several other
mutations that affect the positioning of the mitotic spindle.
Type "a" phenotypes have been reported for cells with
specific mutations in -tubulin (tub2-401; Sullivan and
Huffaker, 1992
) and actin (act1-4; Palmer et al., 1992
), as
well as in dynein (dhc1/dyn1 [Eshel et al., 1993
; Li et al.,
1993
; Yeh et al., 1995
] and jnm1 [McMillan and Tatchell,
1994
]), Act3p/Act5p (Clark and Meyer, 1994
; Muhua et al., 1994
), and Num1p (Kormanec et al., 1991
; Farkasovsky and Kuntzel, 1995
). A type "b" phenotype has
been described for cells with the esp1 mutation (McGrew
et al., 1992
). All of these mutations affect proteins that are
either components of the mitotic spindle or appear to interact with either spindle poles or astral microtubules. Mdm1p may interact with one or more of these components, and evidence for such interaction may emerge from
further genetic analysis of particular Class III alleles.
The intragenic interactions observed among various
mdm1 alleles support a model of Mdm1p assembly into
multimeric structures and suggest that the nature or composition of these multimeric assemblies influences the activity of the Mdm1p network. Intragenic complementation
of some recessive organelle inheritance defects suggests
that functions defective in one allele can sometimes be
supplied by a second allele in trans. In addition, complex genetic phenomena involving various allelic pairs, such as
intragenic suppression and synthetic phenotypes, imply
complex physical interactions between Mdm1p subunits,
such that a domain of one molecule may influence the activity of a nearby region of a partner subunit in a multimeric complex. This model of Mdm1p as a multimeric
complex is consistent with biochemical data indicating the
aggregation of Mdm1p in cellular extracts (McConnell and
Yaffe, 1992) and the assembly of purified Mdm1p into intermediate filament-like structures in vitro (McConnell
and Yaffe, 1993
).
One model for Mdm1p function is that this protein forms a matrix or scaffold-like network in the cytoplasm that is used to position and orient cellular structures. In particular, the spindle pole bodies (SPBs) and mitochondria could be tethered to this scaffolding at particular times during the cell cycle. Changes in mitochondrial distribution or spindle position might involve selective binding and release along the Mdm1p network. A mutation that affects organelle distribution might act either by weakening binding or by preventing appropriate release. For example, if positioning of the mitotic spindle normally involves binding of the maternal SPB to Mdm1p structures and release of the daughter SPB to be carried into the bud, different effects of mdm1 mutations on nuclear position could be explained either by failure to release the daughter SPB (type "a" alleles) or failure to bind the maternal SPB (type "b" alleles). Similarly, mitochondrial distribution and morphology might reflect the binding of the mitochondrial outer membrane or outer membrane-associated proteins to Mdm1p structures. Either loss of such binding or an unregulated or strengthened interaction with Mdm1p could produce the changes in mitochondrial morphology and distribution observed in Class I and Class II mdm1 alleles. The isolation and analysis of allele-specific suppressors, particularly of Class II and Class III alleles, should lead to the identification of proteins that interact with Mdm1p to mediate its functions in nuclear and mitochondrial inheritance.
Received for publication 7 May 1997 and in revised form 13 June 1997.
Please address all correspondence to Dr. Michael P. Yaffe, Department of Biology, 0347, University of California, San Diego, La Jolla, CA 92093-0347. Tel.: (619) 534-4769; Fax: (619) 534-4403; E-mail: myaffe{at}ucsd.eduWe are grateful to Lance Washington for assistance with electron microscopy and to Karen Berger, Randy Hampton, Kelly Shepard, and Mark Nickas for critical reading of the manuscript and their helpful suggestions.
This work was supported by grant GM44614 from the National Institutes of Health and grant MCB-9305338 from the National Science Foundation.
FOA, 5-fluoro-orotic acid; SPB, spindle pole body.
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