Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9148
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Green fluorescent protein (GFP) was used to
tag proteins of the mitochondrial matrix, inner, and
outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated
yeast cells in + ×
0 crosses. When transiently expressed in one of the haploid parents, each of the
marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration
of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged
form of Abf2p, a mtDNA binding protein required for faithful transmission of
+ mtDNA in vegetatively
growing cells, colocalizes with mtDNA in situ. In zygotes of a
+ ×
+ cross, in which there is little mixing
of parental mtDNAs, Abf2p-GFP prelabeled in one
parent rapidly equilibrates to most or all of the
mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In
+ ×
0 crosses,
mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins
distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and
preferential sorting is reduced. These findings argue for
the existence of a segregation apparatus that directs mtDNA to the emerging bud.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MITOCHONDRIA are involved in many essential
metabolic activities and therefore their inheritance is vital for the survival of progeny cells.
However, yeast cells that fail to transmit their wild-type
(+) mitochondrial genomes can still give rise to viable
progeny, although those cells are respiratory deficient.
This essential difference between the inheritance of bulk
mitochondria and mtDNA is readily seen in the yeast Saccharomyces cerevisiae where daughter buds that fail to inherit mitochondria are inviable (Yaffe, 1991
; Berger and
Yaffe, 1996
), but daughter buds that do not receive any
mtDNA (
0 petite mutants) are viable as long as they are
provided with a fermentable carbon source. mtDNA is inherited with great fidelity in wild-type yeast cells grown
under standard laboratory conditions, but different factors, external as well as genotypic, can lead to an instability of the mitochondrial genome. For example,
+ cells
that lack the mitochondrial high mobility group (HMG)1
protein, Abf2p, fail to propagate their mtDNA during
growth on fermentable carbon sources, a defect which
leads to an accumulation of
0 petites in the population
(Diffley and Stillman, 1991
; Megraw and Chae, 1993
;
Megraw et al., 1994
; Zelenaya-Troitskaya et al., 1998
).
Although the analysis of the transmission of mtDNA in vegetatively growing cells has proved useful for identifying genes that function directly or indirectly in mtDNA inheritance, studies with zygotes offer some distinct advantages to dissecting the mechanism of mtDNA inheritance. During zygote maturation, diploid buds can arise from either parental end or from the medial region of the zygote. These budding patterns provide topographical landmarks that facilitate the analysis of sorting of mitochondrial constituents not possible with vegetatively growing cells. For example, zygotes formed from haploid parents that have different mitochondrial genotypes or from parents that have been differentially labeled with mitochondrial marker proteins can be analyzed to determine the fate of these mitochondrial constituents. Such experiments have revealed a number of important features related to the sorting and redistribution of different mitochondrial components during zygote maturation and diploid bud formation.
First, it is clear that mitochondria from each parent fuse
into an extended mitochondrial reticulum in the zygote.
This was suggested from early observations that parental
mtDNAs actively recombine in zygotes (Thomas and
Wilkie, 1968; Dujon et al., 1974
, 1976
) and later from experiments that directly showed the redistribution of prelabeled mitochondrial matrix protein from one parental end of the zygote to the other (Azpiroz and Butow, 1993
). Second, in
+ ×
+ crosses, there is very limited mixing of parental mtDNAs in the fused mitochondrial reticulum of
the zygote; whatever mixing occurs is confined to the neck
region of the zygote where the medial bud emerges. End
buds tend to be genotypically pure for the parental mtDNA of the parent that gave rise to that end of the zygote, whereas medial buds contain both parental (as well
as recombinant) mtDNA molecules (Strausberg and Perlman, 1978
; Zinn et al., 1987
). Thus, mtDNA segregation in
zygotes is not random. Third, in crosses between
+ cells
and certain
petites (cells whose mitochondrial genomes
have suffered large deletions of
+ mtDNA), there can be
a nearly quantitative suppression of the transmission of
+
mtDNAs to the diploid progeny (for reviews see Dujon,
1981
and Piskur, 1994
). Petites exhibiting that property are
called hypersuppressive and their mtDNA genomes characteristically include a ~300-bp element as part of their repeating unit referred to as an ori/rep sequence (Blanc and
Dujon, 1980
, 1982
; de Zamaroczy et al., 1981
).
Finally, the analysis of the sorting of mtDNA and mitochondrial matrix protein markers in zygotes derived from
synchronous matings in + ×
+ and
+ ×
0 crosses
showed that mtDNA movements are independent of the
movements of mitochondrial matrix proteins (Azpiroz and
Butow, 1993
). For example, in zygotes of a
+ ×
+ cross,
where there is little mixing of the parental mtDNAs,
matrix marker proteins rapidly and quantitatively mix
throughout the zygote and into the diploid buds. Similar
conclusions were recently drawn using vital fluorescent
dyes and labeled mtDNA by Nunnari et al. (1997)
. In zygote maturation experiments involving
+ ×
0 crosses, we
observed extensive mixing of
+ mtDNA in the zygote, as
well as mixing of mitochondrial matrix protein markers
(Azpiroz and Butow, 1993
). Significantly, a number of
intermediate zygote forms were detected showing that
mtDNA movements could be well resolved both temporally
and spatially from those of the matrix protein markers.
Here, we describe experiments measuring mtDNA sorting in zygotes relative to additional mitochondrial markers
including inner and outer mitochondrial membrane proteins and a mitochondrial DNA binding protein. These
studies show that mtDNA sorting is independent of the
sorting of the marker proteins of these different mitochondrial compartments. These studies also reveal a remarkable preferential transmission of mtDNA into the medial
diploid bud in + ×
0 crosses, strongly suggesting that
there is an active segregation apparatus that directs
mtDNA into emerging buds.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains and Growth Conditions
The strains used in this study were PSY142 + (MAT
, leu2, lys2, ura3)
and S150-2B
+ (MATa, his3
1, leu2-113, ura3-52, trp1-289).
0 derivatives of these strains were generated by passage of
+ cells on YPD medium containing 25 µg/ml ethidium bromide. PSY142
0 and S150-2B
+
carrying a deletion mutation in the ABF2 gene, called PSY142
abf2
0
and S150-2B
abf2
+, were generated as described below. Cells were
grown at 30°C on YP medium (1% yeast extract and 1% Bactopeptone)
containing either 2% dextrose (YPD) or 2% glycerol (YPGly), or on minimal YNB medium (0.67% yeast nitrogen base without amino acids) containing either 2% dextrose (YNBD) or 2% glycerol (YNBGly), and YNB
medium containing 1% casamino acids and either 2% dextrose (YNBD + cas), 2% glycerol (YNBGly + cas), 2% galactose (YNBGal + cas), 2%
raffinose (YNBR + cas) or 2% galactose and 2% raffinose (YNBGalR + cas) supplemented with nutritional requirements as necessary.
Construction of Green Fluorescent-Fusion Protein Expression Vectors
Plasmid pGAL-CLbGFP was constructed by ligation of a 290-bp BamHI-XhoI fragment containing the 5'-flanking region plus codons 1-52 of the
CIT1 gene, together with a 730-bp XhoI-KpnI fragment containing the
coding regions of a bright green version of green fluorescent protein
(GFP) (bGFP; see below) and a 490-bp KpnI-HindIII fragment containing the 3'- flanking region of CIT1 and ligated into the BamHI-HindIII
site of the CEN-URA3 plasmid pGAL68 (Zelenaya-Troitskaya et al.,
1998). The CIT1 5'-flanking region plus codons 1-52 was generated by
PCR using the primers, 5'-TAAGGGGGATCCTTGCTGTTTAC-3' and
5'-TGCCTTTGCTCGAGTAATTTCAGC-3', and digested with BamHI
and XhoI. The 3'-flanking region of CIT1 was generated by PCR using the primers, 5'-CGAAAGTAGGTACCAAGGAAAATTTG-3' and 5'-GTGACATTAAGCTTGAGGTAAGAAC-3', and digested with KpnI
and HindIII. The bGFP contained three amino acid substitutions, F99S,
M153T, and V163A (our unpublished materials).
To construct plasmid pGAL-YbGFP, a 2.3-kb fragment containing the coding region of the YTA10 gene was generated by PCR using the primers, 5' TTAACGCAGTCTAGAAATAAAGGCATC-3' and 5'-CGTTTTATTTCCTCGAGAATTTGTTGC-3'. The PCR product was digested with XbaI and XhoI. A XbaI-XhoI fragment from pG7GAL-CLbGFP, which was constructed by insertion of a 2.1-kb EcoRI-HindIII fragment from pGAL-CLbGFP ligated into the EcoRI-HindIII site of pGEM-7Zf (+) (Promega Corp., Madison, WI), was replaced with the XbaI-XhoI fragment of the YTA10 gene, yielding pG7-YbGFP. A 3.5-kb XbaI-HindIII fragment from pG7-YbGFP was cloned into the XbaI- HindIII site of pGAL68 to generate pGAL-YbGFP.
To construct plasmid pGAL-bGFPT, a 0.2-kb fragment containing the TOM6 (ISP6/MOM8b) gene was generated by PCR using the primers, 5'-AAATAATTGAAATGCATACGGTATGTTTGC-3' and 5'-AATCTCAACGGTACCAGAACCAAC-3'. The PCR product was digested with NsiI and KpnI. The NH2-terminal in-frame fusion cassette of bGFP, in which the TAG (stop) codon was replaced with a GCA (alanine) codon, was generated by PCR using the plasmid pG7-bGFP as template DNA, and the primers, 5'-ATTTAGGTGACACTATA-3' and 5'-TTCTACGAATATGCATTGTATAGTTCATCC-3'. The PCR product was digested with BamHI and NsiI. A 1-kb BamHI-KpnI fragment from pG3GAL-CLbGFP, which was constructed by insertion of a 2.1-kb EcoRI-HindIII fragment from pGAL-CLbGFP into the EcoRI-HindIII site of pGEM-3Zf(+), was replaced with a BamHI-NsiI fragment (GFP cassette) and a NsiI-KpnI fragment of the TOM6 gene to yield pG3GAL-bGFPT. A 0.8-kb BamHI-HindIII fragment and a 0.6-kb HindIII fragment from pG3GAL-bGFPT were cloned into the BamHI-HindIII site of pGAL68 to generate pGAL-bGFPT.
Plasmid pGAL-Abf2-GFP was constructed as follows: a 1.6-kb BamHI-HindIII fragment consisting of 0.5-kb BamHI-XhoI fragment of the coding region of the ABF2 gene ligated to 0.73-kb XhoI-KpnI fragment of bGFP, ligated to a 0.4-kb KpnI- HindIII fragment containing the 3' flanking region of the ABF2 gene, was cloned into the BamHI-HindIII site of pGAL68 to generate pGAL-Abf2-GFP.
Construction of abf2 Strains
To construct strain PSY142 abf2
0, a 750-bp EcoRI-XhoI fragment containing the 5'- and 3'-flanking regions and entire coding sequence of the
ABF2 gene, was obtained from pRS416/ABF2-GFP (Zelenaya-Troitskaya et al., 1998
), and cloned into the EcoRI-XhoI site of plasmid pBluescript
II KS (+) (Stratagene, La Jolla, CA), creating pBSII-ABF2. The ABF2
coding sequence between the MunI and NheI sites was replaced with a
EcoRI-NheI fragment containing the URA3 gene to generate pBSII-abf2::
URA3. Strain PSY142
0 was transformed with a linearized 1.5-kb EcoRI-XhoI fragment from pBSII-abf2::URA3. S150-2B
abf2
+ was constructed by transforming S150-2B
+ with a linearized 1.6-kb EcoRI fragment from plasmid pAM1A::TRP1 (Diffley and Stillman, 1991
). Disruption in these strains was verified by Southern blotting.
Fractionation of Mitochondria
PSY142 + cells transformed with the various GFP constructs described
above were grown on YNBGalR + cas medium for 16-20 h (OD600 1.2-
1.6) and then converted to spheroplasts using Zymolyase 100T. The
spheroplasts were broken using a loose-fitting Dounce homogenizer and
subjected to differential centrifugation to isolate mitochondria as previously described (Zinser and Daum, 1995
; Newman et al., 1996
). Fractionation of mitochondria, based on published procedures (Pajic et al., 1994
;
Glick, 1995
), was performed as follows: for conversion to mitoplasts, mitochondria (2 mg of protein) were resuspended in 1 ml of hypotonic buffer
(20 mM Hepes-KOH, pH 7.4, containing 0.5% BSA), and incubated for
30 min at 4°C with gentle mixing every 5 min. After 25 min, KCl was
added to a concentration of 80 mM. For control (nonswelling conditions), isolated mitochondria (1 mg of protein) were incubated in 1 ml of isotonic
buffer (0.6 M sorbitol, 20 mM Hepes-KOH, pH 7.4, containing 0.5%
BSA), and as indicated in the Results, treated with proteinase K at a concentration of 50 µg/ml for 20 min on ice, followed by a 10-min incubation
on ice with 1 mM of PMSF. Mitochondria and mitoplasts were reisolated
by centrifugation for 10 min at 12,000 g. Mitochondria were resuspended
in 1 ml of SDS sample buffer. For carbonate extraction, mitoplasts were
resuspended in 1 ml of extraction buffer (0.1 M Na2CO3 and 1 mM
PMSF), and incubated for 30 min. For sonication, mitoplasts were resuspended in 1 ml of sonication buffer (0.1 M NaCl, 20 mM Hepes-KOH, pH
7.4, and 1 mM PMSF) and subjected to three rounds of freeze-thaw at
80°C, followed by sonicating at 70 W for 30 times at 1-s each with a sonifier (model 450; Branson Ultrasonics Corp., Danbury, CT). The samples
from carbonate extraction and sonication were centrifuged (Sorvall Ultra
80; Dupont, Newtown, CT) at 226,000 g for 60 min. The pellet fractions
were resuspended in 1 ml of SDS sample buffer. All samples were analyzed by SDS-PAGE and Western blotting, probed with a polyclonal antibody of GFP (Clontech Laboratories, Inc., Palo Alto, CA), and detected
using a goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad Laboratories, Inc., Hercules, CA) and the enhanced chemiluminescence reagents
(Amersham Corp., Arlington Heights, IL).
Induction of Protein-GFP Fusions
PSY142 + or
0 cells transformed with GFP chimeric genes under control
of the GAL1-10 promoter in the pGAL68 derivatives were grown on
YNBR +cas medium to mid-log phase, inoculated into YNBGalR + cas
medium and grown to induce the synthesis of the protein-GFP fusions
(12-18 h for CS1-GFP, Yta10p-GFP, and GFP-Tom6p; 45-60 min for
Abf2p-GFP). These different induction times were chosen from preliminary experiments to optimize detection of each GFP fusion protein. S150-2B
abf2
+ transformants precultured on YNBGly +cas medium to mid-log
phase, were transferred to YNBGal + cas medium, and then incubated
for 2 h to induce the synthesis of CS1-GFP. Cells were collected by centrifugation before synchronized mating.
Sorting Analysis of mtDNA and Protein-GFP Fusion in Zygotes
Synchronized mating was performed as previously described (Azpiroz and
Butow, 1995). For fixation, cells from mating mixtures at each time point
(1-4 or 6 h) were resuspended in 1 ml of 3.7% formaldehyde solution and
incubated for 1 h at 30°C. After three washes in water, the cells were
stained by brief incubation in 1 µg/ml 4',6-diamino-2-phenylindole
(DAPI) in water followed by three washes in water. The samples were resuspended in 0.25% low melting agarose solution prewarmed at 37°C,
placed on a microscope slide protected by a coverslip, and then kept for 10 min at 4°C. Zygotes were scored according to the previous procedures
(Azpiroz and Butow, 1993
, 1995
). At least 100 zygotes were scored for
each time point presented in Figs. 4-7, 9, and Table I.
|
|
|
|
|
|
|
Microscopy and Image Analysis
The samples were observed using a Leica microscope (model DMRXE; Deerfield, IL) equipped for an HBO 100 W/2 mercury arc lamp, an X100 Plan-Apochromat objective, and epifluorescence with the following filter sets: (a) 340-380-nm band-pass excitation filter; (b) 400-nm dichroic reflector; (c) \>425-nm long-pass emission filter for DAPI, and (d) 450-490, 510, and \>515 for GFP. Differential interference contrast and fluorescence images were captured with a color-chilled three charge-coupled device camera system (model C5810; Hamamatsu Phototonics, Bridgewater, NJ) and then processed using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mitochondrial Marker Protein-GFP Fusions
To examine the sorting of mitochondrial components during zygote maturation, we prelabeled a 0 strain with a mitochondrial marker protein of interest and then synchronously mate those cells to a
+ strain lacking the marker
protein; fluorescence microscopy is then used to monitor
the sorting of the marker protein and DAPI-stained mtDNA during the course of zygote maturation and diploid bud formation. In this study we introduce a new terminology for describing these crosses. Where both markers (i.e., mtDNA and a protein-GFP fusion) originate in
the same parent, the experiment is referred to as a cis
cross; where each parent introduces a different marker to the zygote, it is referred to as a trans cross. The
+ ×
0
trans crosses are particularly useful for revealing how mitochondrial constituents sort, since the initial separation of
the mitochondrial marker protein and mtDNA at opposite
ends of the newly formed zygote allows a clear-cut time
and spatial resolution of the sorting process (Azpiroz and
Butow, 1993
).
To extend this analysis to other mitochondrial compartments, we have constructed a series of gene fusions that
would direct GFP to the mitochondrial matrix, the inner
and outer mitochondrial membranes, and mtDNA. As
shown in Fig. 1 A, these gene fusions were constructed to
encode chimeric proteins between GFP and (a) 52 amino
acids of the NH2-terminal region of the matrix protein, citrate synthase 1 (CS1); (b) the COOH terminus of the full-length inner membrane protease subunit, Yta10p (Tauer
et al., 1994; Arlt et al., 1996
; Guelin et al., 1996
); (c) the
NH2 terminus of outer membrane protein, Tom6p (Isp6p),
a component of a complex of the outer membrane, protein
import apparatus (Kassenbrock et al., 1993
; Alconada et al.,
1995
); and (d) the COOH terminus of the HMG protein,
Abf2p, that binds to mtDNA (Diffley and Stillman, 1992
;
Newman et al., 1996
; Zelenaya-Troitskaya et al., 1998
). Expression of each of these gene fusions was placed under
the control of the GAL1-10 promoter in the plasmid
pGAL68 and transformed into PSY142
+ cells. All of
these fusion proteins were found to be stable under the
conditions of the zygote sorting experiments, and their
mRNAs were essentially undetectable by Northern blot
analysis of total RNA isolated from cells immediately following the 2-h incubation in the dextrose medium used before the initiation of synchronous mating (data not shown).
|
To determine whether these fusion proteins are targeted
to mitochondria, cells from log-phase cultures of the transformants grown in YNBGalR + cas medium were examined by epifluorescence microscopy. Fig. 1 B shows that,
with the exception of Abf2p-GFP, expression of each of
the GFP fusion constructs results in a morphological pattern characteristic of the tubular mitochondrial network
located near the periphery of the cell (Fig. 1 B) (Hoffman
and Avers, 1973). In contrast, Abf2p-GFP, which complements the mtDNA instability phenotype of a
abf2 mutant strain (data not shown), shows a distinctly punctate
pattern coincident with that of DAPI-stained mtDNA. We showed recently that this Abf2p-GFP fluorescence pattern reflects the association of Abf2p-GFP with mtDNA
in vivo (Zelenaya-Troitskaya et al., 1998
). In that report,
we demonstrated that a GFP derivative of a mutant form
of Abf2p containing mutations of key amino acid residues
in each of the HMG boxes that inhibit DNA binding in vitro had an in vivo fluorescence pattern that more closely
resembled that of a matrix protein than the punctate pattern seen for the wild-type Abf2p-GFP derivative. Moreover, mtDNA nucleoids isolated according to the procedure
described in Newman et al. (1996)
from cells expressing
Abf2p-GFP contained this fusion protein at levels comparable to that of endogenous Abf2p (data not shown).
With the exception of Abf2p-GFP, no conclusions can
be drawn from the fluorescence patterns alone about the
localization of the GFP fusion proteins within specific mitochondrial compartments. Therefore, to verify that each
GFP fusion protein is localized to the intended mitochondrial compartment, mitochondria were isolated from
strain PSY142 + expressing each of the GFP fusion proteins and analyzed biochemically to determine the protein
localization. In these experiments, the GFP fusion proteins were detected by Western blotting using anti-GFP antiserum. Fig. 2 shows that when mitochondria were
treated with proteinase K, the CS1- and Yta10p-GFP fusion proteins were largely resistant to proteolysis, whereas
the GFP-Tom6p fusion protein was completely digested,
consistent with the known localization and topology of
Tom6p in the outer mitochondrial membrane (Kassenbrock et al., 1993
). Next, crude mitoplasts were prepared
by swelling of isolated mitochondria as described in Materials and Methods, and treated either with Na2CO3, pH
11.5, to release soluble proteins, or sonicated; in both
cases, the treated mitoplasts were separated by centrifugation into soluble and pellet fractions. Fig. 2 shows that
CS1-GFP was released into the supernatant fraction by
treatment with Na2CO3 as well as by sonication. The incomplete release of the CS1-GFP fusion protein by sonication was due to some trapping of the protein by vesicle
resealing, which we have verified occurs in parallel experiments with native mitochondrial matrix proteins (data not
shown). In contrast to these results, GFP-Tom6p and Yta10p-GFP remained associated with the pellet fractions
of the treated mitoplasts. The presence of some signal for
GFP-Tom6p is likely due to contamination of the crude
mitoplast preparations with outer membrane. Taken together, these results demonstrate that each of these fusion
proteins is localized to the intended mitochondrial compartment.
|
Sorting Patterns of Mitochondrial Matrix and
Membrane Proteins in + ×
0 Crosses
We previously identified four prominent zygote types,
shown diagramatically in Fig. 3, that were evident in + ×
0 trans crosses when the
0 cells were prelabeled with a
mitochondrial matrix protein marker (Azpiroz and Butow,
1993
). The type U (unmixed) form, in which the mitochondrial protein marker and mtDNA were separated in opposite ends of the zygote, appeared immediately upon zygote
formation. This zygote form gradually disappeared with
time and was replaced by various intermediate forms,
leading eventually to the type M (mixed) zygote in which
the marker protein and mtDNA were colocalized and distributed throughout the zygote and into the emerging diploid buds. An unexpected and novel zygote type that appeared early in zygote maturation was the A (asymmetric)
form, in which the matrix marker protein had quantitatively translocated from the
0 end of the zygote through
the fused mitochondrial reticulum into the
+ end, well before any movement of mtDNA. Although A form zygotes may not be an obligatory intermediate in the sorting process from U to M zygotes, the mechanism by which that
novel zygote type arises remains a mystery, and their presence provides a dramatic illustration of the temporal and
spatial resolution of matrix protein and mtDNA within the
fused mitochondrial reticulum of the zygote.
To extend these analyses to proteins in other mitochondrial compartments, we followed the time course of sorting of the various GFP fusion proteins and mtDNA in + ×
0 trans crosses. The fusion proteins were first expressed in
PSY142
0 cells grown in YNBGalR + cas medium, before
initiation of synchronous matings to S150-2B
+ cells (refer to Materials and Methods). After mating, aliquots of
the cells were removed at 1-h intervals and examined by
epifluorescence microscopy for the distribution of the
GFP fusion proteins and mtDNA by DAPI staining. Representative micrographs of the various zygote types we detected in these crosses are presented in Figs. 4-6, panels A.
In Fig. 4-6, panels B, accompanying each micrograph is a
plot of the kinetics of appearance and disappearance of
the various zygote forms detected.
The sorting patterns observed in the cross with the CS1-
GFP matrix marker (Fig. 4) are essentially the same as we
previously described for native CS1 and for the chimeric
protein OTCase-dihydrofolate reductase (DHFR) (Azpiroz
and Butow, 1993). The population of unmixed zygotes, in
which CS1-GFP is in the
0 end and mtDNA is in the
+
end, disappears with time, and by 4 h after zygote formation is replaced almost entirely by the mixed, M-type zygote in which CS1-GFP and mtDNA are distributed to all
parts of the cell including the diploid buds. During the
course of sorting from the U to M forms, we detected the
same two intermediate zygote forms detected previously.
One is the P-type zygote, which appears within 1 h of zygote formation, and reaches a maximum of ~50% of the
zygote population by 2 h. In this zygote type some of the
CS1-GFP present initially in the
0 end of the zygote has
moved into the
+ end before any appreciable movement
of mtDNA. The second intermediate zygote type is the
novel A form, which reaches a maximum at 2 h, and accounts for roughly 10% of the zygote population.
Figs. 5 and 6 show the sorting of the inner and outer membrane marker fusion proteins, Yta10p-GFP and GFP- Tom6p, respectively. Similar to matrix protein markers, both of these membrane marker proteins equilibrate throughout the fused mitochondrial reticulum of the zygote and into the diploid buds. There are, however, two notable differences between the sorting patterns of these membrane marker proteins and those of the matrix markers. First, we have never detected any A form zygotes in crosses involving Yta10p-GFP and Tom6p-GFP. Second, the kinetics of sorting of both of the membrane marker proteins is slower than that observed for CS1-GFP (compare Fig. 5 B with Figs. 6 B and 7 B), evident as an ~1 h delay in the half-time for the disappearance of the U-type zygotes and the time required for appearance of the maximum number of P-type zygotes. These experiments show that, like proteins in the matrix, inner and outer membrane proteins can translocate through the fused mitochondrial reticulum.
Sorting Pattern of Abf2p-GFP in a +×
+ Cross
In + ×
+ crosses it has long been clear that there is only a
very limited mixing of parental mitochondrial genomes,
which is confined to the middle or neck region of the zygote where medial buds appear (Strausberg and Perlman,
1978
; Zinn et al., 1987
; Azpiroz and Butow, 1993
). One
possibility is that mtDNA, as well as proteins associated
with it, represents a compartment of the mitochondrion
that exhibits only limited mixing during zygote maturation. To test this notion, we used Abf2p-GFP, which is a functional form of this protein that colocalizes with
mtDNA, as a marker for the mtDNA compartment and
tested how this protein sorts in zygotes of a
+ ×
+ cross.
Possible outcomes are that either Abf2p-GFP, prelabeled in one of the parents, remains with the mtDNA of that
parent, or that it is capable of equilibrating among all of
the mtDNA molecules in the zygote.
We performed a + ×
+ cross in which Abf2p-GFP was
prelabeled in PSY142
+ cells in the same manner as described above for prelabeling cells with markers for other
mitochondrial membrane compartments, and then synchronously mated to S150-2B
+ cells. The results of this
experiment (Fig. 7) show that Abf2p-GFP rapidly equilibrates throughout the zygote and into the diploid bud.
Moreover, the half-time for disappearance of the U form
zygotes is roughly 1 h, similar to that of CS1-GFP U form
zygotes (refer to Fig. 4 B). Thus, despite the fact that
+
parental mtDNAs do not mix in
+ ×
+ crosses, Abf2p-
GFP rapidly redistributes among all of the mtDNA in the
zygote. This finding also indicates that although Abf2p- GFP may be a useful marker for mtDNA in static experiments, it is not useful for examining the dynamics of
mtDNA movements in this experiment.
Preferential Transmission of mtDNA into Medial Diploid Buds
During the scoring of the various zygote forms generated
in the trans crosses described thus far, we noticed that in
some cases mtDNA appeared to have been distributed
preferentially from the + end of the zygote into the diploid bud. To examine this potentially important aspect of
mtDNA sorting in more detail, we have performed cis
+ ×
0 crosses in which the various mitochondrial marker proteins were transiently expressed in the
+ parent before
mating with
0 cells. In this cis configuration, both mtDNA
and the mitochondrial marker protein are initially present
in one end of the newly formed zygote, allowing us to observe how these components distribute with time through
the fused mitochondrial reticulum to different regions of
the zygote and the emerging diploid buds. One possible outcome is that both mtDNA and the marker protein distribute randomly to the other end of the zygote and to the
emerging bud; alternatively, the protein and mtDNA may
sort differently. In these experiments we have examined
zygotes that contain a medial bud between one-third and
two-thirds the size of a mature diploid bud. These zygotes, which are most abundant between 2-3 h after mating, have
not yet fully equilibrated these mitochondrial components
and are thus optimal to detect intermediates in the sorting
process (Table I).
Immediately following zygote formation we observed
the expected unmixed, unbudded cis zygote type containing mtDNA and the mitochondrial GFP marker protein
together in one end of the cell (Fig. 8). A survey of zygotes
with a medial bud scored after 2 and 3 h of mating reveals
three major zygote types (I-III), representative examples
of which are shown in Fig. 8; a quantification of the relative abundance of these zygote types in crosses involving each of the marker proteins is presented in Table I. Type I
zygotes are those in which the protein marker and
mtDNA have fully equilibrated throughout the zygote and
into the diploid buds. These are equivalent to the M form
zygotes that are generated in the + ×
0 trans crosses described earlier, and they represent between one-fourth and one-fifth of the zygote population for each of the
marker proteins (Table I). Type II zygotes are those in
which both mtDNA and the marker protein have sorted
together and predominately into the diploid bud; they represent the least abundant of the zygote types observed.
|
By far the most abundant zygote form is type III in
which mtDNA appears preferentially in the diploid bud,
whereas the protein marker has equilibrated throughout
the zygote as well as into the diploid bud. This unique sorting of mtDNA is particularly evident in zygotes expressing
CS1-GFP where the type III zygotes account for 81% of
the total zygotes in the population (Table I). Type III zygotes are also the majority type with the inner and outer
mitochondrial membrane markers, although they represent a somewhat smaller fraction of the total zygote population in those crosses. The reduction in type III zygotes
with the membrane markers could be accounted for by a
slower mixing of those markers relative to the matrix
marker, and a slight increase in the preferential sorting of
those membrane marker proteins. An important conclusion from these experiments is that mtDNA appears to
sort from the + end of the zygote preferentially into the
emerging diploid bud, whereas the marker proteins, and
especially the matrix marker, appear to equilibrate more
or less randomly in the zygote with little preference for the
medial diploid bud.
Sorting of mtDNA Is Altered in a Homozygous
abf2 Cross
The mitochondrial HMG protein, Abf2p, is required for
the maintenance of + mtDNA in cells grown on medium
containing glucose as the carbon source, whereas
+
mtDNA can be maintained indefinitely in such cells if they
are grown on medium containing a nonfermentable carbon source. In those cells, mtDNA nucleoids visualized by
DAPI staining of cells appear more disorganized than nucleoids in wild-type cells, and some polypeptides present
in isolated nucleoids are depleted or missing compared
with nucleoids from wild-type cells (Newman et al., 1996
).
Those findings suggested a relationship between the organization of mtDNA and its stability. Given the results presented thus far, it was of interest to determine whether the
sorting of mtDNA is altered in zygotes that lack Abf2p.
To examine this question, a + ×
0 cis cross was performed with the CS1-GFP matrix marker using derivatives of strains PSY142 and S150-2B in which the ABF2
gene was deleted. As with
abf2 haploid cells, mtDNA is
also unstable in diploids derived from these cells (data not
shown). The
+
abf2 derivative was prelabeled with CS1-
GFP in exactly the same way as detailed in the preceding
section for its ABF2 counterpart, except that the cells were
maintained on YNBGly + cas medium before induction of
CS1-GFP. In preliminary experiments carried out with crosses between these
abf2 strains, we observed that
mtDNA sorting appeared to be slower than in the control,
ABF2 × ABF2 crosses. Therefore, we extended the time
course of the analysis to examine zygotes with medial buds
to include 4- and 5-h time points in addition to scoring at
the 2- and 3-h intervals as before. As shown in Fig. 8 and
summarized in Table II, in addition to the zygote types (I-
III) found in the homozygous ABF2 cross, a new zygote
type (IV) is detected in this
abf2 cross in which the CS1-
GFP marker has fully equilibrated in the zygote and into the diploid bud, but mtDNA has not yet moved out of the
+ end of the zygote. Type IV zygotes were never observed in ABF2 crosses. This delay in sorting of mtDNA
compared with wild-type crosses accounts, in part, for the
smaller fraction of type III zygotes observed at the earlier
time points. Throughout the time course of the analysis,
the fraction of type III zygotes never reaches that observed with the CS1-GFP marker in the wild-type cross
(refer to Table I), indicating that the absence of Abf2p not
only delays the onset of mtDNA equilibration, but also reduces the preferential sorting into the medial diploid bud.
|
To show directly that there is a delay in mtDNA sorting
in the abf2 cross, we carried out a side by side comparison of the kinetics of appearance of M form zygotes in the
abf2 cross with that of the wild-type (ABF2) cross. The
data of Fig. 9 show that the appearance of M-type zygotes
in the
abf2 cross is delayed by ~1 h relative to the ABF2
control cross. What is clear from these experiments is that
the absence of Abf2p results in a reduction of the type III
zygotes showing preferential transmission of mtDNA into
the diploid bud because that population is replaced with the type IV zygotes.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this paper we show that the sorting of GFP marker proteins located in four different mitochondrial compartments can be spatially and temporally resolved from the
sorting of mtDNA in zygotes of synchronously mated
cells. In addition, we show that in cis crosses, mtDNA, in
contrast to the marker proteins, is first directed preferentially to the medial diploid bud before fully equilibrating
to the 0 end of the zygote. To carry out these experiments, we constructed a set of gene fusions designed to
target GFP to the matrix, the inner, and outer mitochondrial membranes, as well as to mtDNA. Biochemical experiments verified the specific mitochondrial location of
the matrix and membrane-bound GFP fusion proteins,
whereas the localization of the Abf2p-GFP fusion protein
to mtDNA was evident from direct fluorescence microscopic analysis, which showed a punctate staining pattern
identical to that of DAPI-stained mtDNA. In a previous
study (Zelenaya-Troitskaya et al., 1998
), we demonstrated
that the punctate staining pattern of Abf2p-GFP was dependent on the ability of the protein to bind to DNA,
since an Abf2p-GFP fusion protein derived from a mutant
form of Abf2p with mutations of the two HMG boxes that
markedly reduced DNA binding activity in vitro, showed
an in vivo staining pattern that was more typical of that
observed with a mitochondrial matrix protein than the
punctate staining of mtDNA.
The sorting pattern of the CS1-GFP marker in zygotes
from a + ×
0 trans cross, in which the
0 parent was
prelabeled with the marker protein, agrees well with the
pattern previously described using indirect immunofluorescence methods to detect endogenous CS1 and a foreign
protein (DHFR) targeted to the mitochondrial matrix
(Azpiroz and Butow, 1993
). In particular, we observed the
appearance of the novel A form zygote where, within 2 h
after zygote formation, the marker protein initially present
in the
0 end of the cell quantitatively translocated to the
+ end before any significant movement of
+ mtDNA.
We do not yet understand the mechanism by which A
form zygotes are generated or why this unusual intermediate form is only observed in zygotes from
+ ×
0 crosses,
independent of whether the
+ parent is respiratory competent or not (Azpiroz and Butow, 1993
). The only other
instance where we failed to observe A form zygotes using
a matrix marker protein for the sorting analysis was in
+ ×
0 trans crosses between
abf2 parental strains (Zelenaya-Troitskaya et al., 1998
). A further consideration of the effects of the absence of Abf2p in the sorting patterns in zygotes is discussed below.
Both the inner and outer mitochondrial membrane
marker proteins, Yta10p-GFP, and GFP-Tom6p, respectively, translocated with very similar kinetics through the
fused mitochondrial reticulum; however, their rates of
equilibration were slower than that observed for CS1-
GFP. This is not an unexpected result, since the lateral diffusion of proteins through membranes has generally been
observed to be slower than diffusion of nonmembrane
proteins. The observed diffusion coefficients for membrane proteins can, however, vary greatly depending on
the protein or protein complex, the particular membrane
and whether there is an interaction between the membrane protein and components such as cytoskeletal elements (Jacobson et al., 1987). For mitochondria, the diffusion properties of respiratory chain complexes of the inner
mitochondrial membrane have been studied (Chazotte
and Hackenbrock, 1991
). Those experiments showed that
there were no significant differences in diffusion coefficients of the inner membrane complexes examined and
that the observed rates of diffusion were insensitive to
membrane folding or the environment immediately adjacent to the membrane. We can conclude from the present
studies that when parental mitochondria fuse in zygotes,
mitochondrial membrane as well as matrix proteins can
exchange throughout the extended mitochondrial reticulum. Finally, besides the slower equilibration rate of
Yta10p-GFP and GFP-Tom6p, the other notable difference between the sorting pattern of these membrane proteins and matrix markers is the absence of any detectable
A form zygotes. Since we do not know the mechanism by
which A form zygote arise in crosses with the matrix markers, it is difficult to speculate on why that novel zygote
type is not observed with the membrane markers.
The sorting of Abf2p-GFP was analyzed somewhat differently than the other mitochondrial marker proteins in
that it was transiently expressed in one of the parents of a
+ ×
+ cross. Because the parental mtDNAs in zygotes
from
+ ×
+ crosses do not mix to any appreciable extent
during zygote maturation (Strausberg and Perlman, 1978
;
Zinn et al., 1987
; Azpiroz and Butow, 1993
; Nunnari et al.,
1997
), it was of particular interest to know whether
Abf2p-GFP would remain with the parental mtDNA or
partition to all of the mtDNA nucleoids of the zygote. We
found that Abf2p-GFP rapidly equilibrated throughout
the zygote, eventually colocalizing with the
+ mtDNA of
the unlabeled parent, with kinetics similar to that of CS1-GFP. These results indicate that even though parental
+
mtDNAs do not mix in the zygote, the parental mtDNA
compartments are nevertheless fully accessible to each
other in the fused mitochondrial reticulum.
The finding of preferential transmission of mtDNA to
the medial diploid bud provides the strongest evidence yet
that mtDNA inheritance is not stochastic. The preponderance of type III zygotes in the + ×
0 cis crosses of Table I
is particularly significant in that both mtDNA and the
marker protein, present initially in the
+ end of the zygote, have in principle the same opportunity to segregate
through the mitochondrial reticulum to the
0 end of the
zygote as well as to the medial diploid bud. Yet, for the
majority of zygotes, mtDNA first appears in the bud,
whereas the marker protein distributes more or less randomly between the bud and the
0 end of the zygote. This
clear-cut distinction between preferential versus random
transmission of mtDNA cannot be made for
+ ×
+
crosses, since the well-documented restriction of mtDNA
movement within the body of the zygote means that in either circumstance only the medial buds would receive a
sample of both parental mtDNAs (Strausberg and Perlman, 1978
; Zinn et al., 1987
; Nunnari et al., 1997
).
In zygotes lacking Abf2p, the process of mtDNA sorting
is altered in two significant ways: first, its movement out of
the + end of the zygote is delayed, as evident by type IV
zygotes where the DNA remains in the
+ end but the
marker protein has equilibrated throughout the zygote. Second, the preferential sorting of mtDNA to the medial
bud is reduced. Since in zygotes, mtDNA mixing and sorting to buds are important parameters that can affect the
observed frequency of mtDNA recombination in crosses
(Strausberg and Perlman, 1978
; Zinn et al., 1987
), these alterations in sorting probably contribute to the observation
that recombination between mtDNA markers is suppressed in homozygous
abf2 crosses (Zelenaya-Troitskaya et al., 1998
). However, we have recently found
that Abf2p also affects the level of mtDNA recombination
intermediates (Holliday junctions) (MacAlpine et al.,
1998
), suggesting that this protein has a direct role in recombination.
In the zygote system we have used, it is useful to draw a
distinction between sorting within the fused mitochondrial
reticulum of the zygote itself, and sorting from the zygote
to the emerging diploid bud. In the latter, sorting would be
accompanied by a directed and continuous transfer of mitochondria to the bud whereas in the former, sorting
would occur within the mitochondrial reticulum of the zygote and be subject only to intracellular mitochondrial dynamics, e.g., fusion and fission events. For bulk mitochondrial inheritance, there is now considerable evidence that
the transmission of mitochondria from mother to daughter
cell is an active, regulated process requiring a number of
gene products (McConnell et al., 1990; McConnell and
Yaffe, 1992
; Sogo and Yaffe, 1994
; Hermann et al., 1997
).
There is, in addition, evidence to suggest that mitochondrial organization and inheritance in yeast is linked to the
actin cytoskeleton (Drubin et al., 1993
; Lazzarino et al., 1994
; Simon et al., 1995
; Smith et al., 1995
). There is much
less information on how mitochondrial dynamics, and particularly fusion and fission events, are controlled. Recently, a predicted transmembrane GTPase encoded by
the fuzzy onion gene in Drosophila has been implicated in
mediating mitochondrial fusion during Drosophila spermatogenesis (Hales and Fuller, 1997
). It will be of great interest to learn whether yeast require a similar type of protein for mitochondrial fusion.
The process that accounts for the preferential sorting of
mtDNA to the zygotic bud may also account for its faithful
segregation in vegetatively growing cells; that is, mtDNA
segregation might be firmly coupled to mitochondrial
transmission. It is intriguing that the absence of Abf2p in
zygotes results in a decrease, but not in an elimination, of
the preferential sorting of mtDNA to the bud, and that
mtDNA transmission is compromised but not blocked altogether in abf2 cells (In
abf2 cells the rate of mtDNA
loss is significantly slower than the division time [Zelenaya-Troitskaya et al., 1998
]). Perhaps in the absence of
Abf2p, mtDNA-protein complexes are altered such that
mtDNA is less able to engage the putative segregation apparatus, resulting in an uncoupling between mitochondrial
transmission and mtDNA segregation. Studies are currently underway to identify proteins that would both interact with mtDNA and function in the transmission process.
Our finding of directed movements of mtDNA warrants
drawing an analogy to the mitotic apparatus for chromosomal segregation that insures each cell receives its full
chromosome complement during cell division. Although it
is unlikely that mtDNA segregation will turn out to be as
complicated or as precise as chromosome segregation in
mitosis and meiosis, accumulating evidence suggests that it will not be as simple as random sorting.
![]() |
Footnotes |
---|
Received for publication 5 March 1998 and in revised form 26 June 1998.
Address all correspondence to Ronald A. Butow, Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9148. Tel.: (214) 648-1465. Fax: (214) 648-1488. E-mail: butow{at}swmed.eduWe are grateful to S. Newman (University of Texas Southwestern, Dallas, TX) for making available some of the ABF2 constructs used and for advice and helpful suggestions during the course of this study. We thank other members of the Butow/Perlman lab for helpful discussions.
This work was supported by grants from the National Institutes of Health (GM 33510) and The Robert A. Welch Foundation (I-0642).
![]() |
Abbreviations used in this paper |
---|
CS1, citrate synthase 1; DAPI, 4',6-diamino-2-phenylindole; GFP, green fluorescent protein; HMG, high mobility group; Tom, translocase outer membrane.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Alconada, A., M. Kübrich, M. Moczko, A. Hönlinger, and N. Pfanner. 1995. The mitochondrial receptor complex: the small subunit Mom8b/Isp6 supports association of receptors with the general insertion pore and transfer of preproteins. Mol. Cell. Biol 15: 6196-6205 [Abstract]. |
2. | Arlt, H., R. Tauer, H. Feldmann, W. Neupert, and T. Langer. 1996. The YTA10-12 complex, an AAA protease with chaperone-like activity in the inner membrane of mitochondria. Cell 85: 875-885 |
3. | Azpiroz, R., and R.A. Butow. 1993. Patterns of mitochondrial sorting in yeast zygotes. Mol. Biol. Cell 4: 21-36 [Abstract]. |
4. | Azpiroz, R., and R.A. Butow. 1995. Mitochondrial inheritance in yeast. Methods Enzymol 260: 453-466 |
5. | Berger, K.H., and M.P. Yaffe. 1996. Mitochondrial distribution and inheritance. Experentia 52: 1111-1116 . |
6. | Blanc, H., and B. Dujon. 1980. Replicator regions of the yeast mitochondrial DNA responsible for suppressiveness. Proc. Natl. Acad. Sci. USA 77: 3942-3946 [Abstract]. |
7. | Blanc, H., and B. Dujon. 1982. Replicator regions of the yeast mitochondrial DNA active in vivo and in yeast transformants. In Mitochondrial Genes. P. Slonimski, P. Borst, and G. Attardis, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 279-294. |
8. |
Chazotte, B., and
C.R. Hackenbrock.
1991.
Lateral diffusion of redox components in the mitochondrial inner membrane is unaffected by inner membrane folding and matrix density.
J. Biol. Chem
266:
5973-5979
|
9. | de Zamaroczy, M., R. Marotta, G. Faugeron-Fonty, R. Goursot, M. Mangin, G. Baldacci, and G. Bernardi. 1981. The origins of replication of the yeast mitochondrial genome and the phenomenon of suppressivity. Nature 292: 75-78 |
10. | Diffley, J.F., and B. Stillman. 1991. A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria. Proc. Natl. Acad. Sci. USA 88: 7864-7868 [Abstract]. |
11. |
Diffley, J.F.X., and
B. Stillman.
1992.
DNA binding properties of an HMG1-
related protein from yeast mitochondria.
J. Biol. Chem
267:
3368-3374
|
12. | Drubin, D.G., H.D. Jones, and K.F. Wertman. 1993. Actin structure and function-roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site. Mol. Biol. Cell 4: 1277-1294 [Abstract]. |
13. | Dujon, B. 1981. Mitochondrial Genetics and Functions. In The Molecular Biology of the Yeast Saccharomyces. J.N. Strathern, E.W. Jones, and J.R. Broachs, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 505-635. |
14. | Dujon, B., M. Bolotin-Fukuhara, D. Coen, J. Deutsch, P. Netter, P.P. Slonimski, and L. Weill. 1976. Mitochondrial genetics. XI. Mutations at the mitochondrial locus omega affecting the recombination of mitochondrial genes in Saccharomyces cerevisiae. Mol. Gen. Genet 143: 131-165 |
15. |
Dujon, B.,
P.P. Slonimski, and
L. Weill.
1974.
Mitochondrial genetics. IX. A
model for the recombination and segregation of mitochondrial genes in Saccharomyces cerevisiae.
Genetics
78:
415-437
|
16. | Glick, B.S.. 1995. Pathways and energetics of mitochondrial protein import in Saccharomyces cerevisiae. Methods Enzymol 260: 224-231 |
17. | Guelin, E., M. Rep, and L.A. Grivell. 1996. Afg3p, a mitochondrial ATP- dependent metalloprotease, is involved in degradation of mitochondrially- encoded Cox1, Cox3, Cob, Su6, Su8 and Su9 subunits of the inner membrane complexes III, IV and V. FEBS (Fed. Eur. Biochem. Soc.) Lett 381: 42-46 . |
18. | Hales, K.G., and M.T. Fuller. 1997. Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90: 121-129 |
19. |
Hermann, G.J.,
E.J. King, and
J.M. Shaw.
1997.
The yeast gene, MDM20, is
necessary for mitochondrial inheritance and organization of the actin cytoskeleton.
J. Cell Biol
137:
141-153
|
20. | Hoffman, H., and C.J. Avers. 1973. Mitochondrion of yeast: ultrastructural evidence for one giant, branched organelle per cell. Science 181: 749-751 |
21. | Jacobson, K., A. Ishihara, and R. Inman. 1987. Lateral diffusion of proteins in membranes. Annu. Rev. Physiol 49: 163-175 |
22. | Kassenbrock, C.K., W. Cao, and M.G. Douglas. 1993. Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex. EMBO (Eur. Mol. Biol. Organ.) J 12: 3023-3034 [Abstract]. |
23. | Lazzarino, D.A., I. Boldogh, M.G. Smith, J. Rosand, and L.A. Pon. 1994. Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity. Mol. Biol. Cell 5: 807-818 [Abstract]. |
24. |
MacAlpine, D.M.,
P.S. Perlman, and
R.A. Butow.
1998.
The high mobility
group protein, Abf2p, influences the level of yeast mitochondrial DNA recombination intermediates in vivo.
Proc. Natl. Acad. Sci. USA
95:
6739-3743
|
25. | McConnell, S.J., L.C. Stewart, A. Talin, and M.P. Yaffe. 1990. Temperature-sensitive yeast mutants defective in mitochondrial inheritance. J. Cell Biol 111: 967-976 [Abstract]. |
26. | McConnell, S.J., and M.P. Yaffe. 1992. Nuclear and mitochondrial inheritance in yeast depends on novel cytoplasmic structures defined by the MDM1 protein. J. Cell Biol 118: 385-395 [Abstract]. |
27. |
Megraw, T.L., and
C.B. Chae.
1993.
Functional complementarity between the
HMG1-like yeast mitochondrial histone HM and the bacterial histone-like
protein HU.
J. Biol. Chem
268:
12758-12763
|
28. | Megraw, T.L., L.R. Kao, and C.B. Chae. 1994. The mitochondrial histone HM: an evolutionary link between bacterial HU and nuclear HMG1 proteins. Biochimie 76: 909-916 |
29. |
Newman, S.M.,
O. Zelenaya-Troitskaya,
P.S. Perlman, and
R.A. Butow.
1996.
Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain
of Saccharomyces cerevisiae that lacks the mitochondrial HMG-box protein,
Abf2p.
Nucl. Acids Res
24:
386-393
|
30. | Nunnari, J., W.F. Marshall, A. Straight, A. Murray, J.W. Sedat, and P. Walter. 1997. Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA. Mol. Biol. Cell 8: 1233-1242 [Abstract]. |
31. | Pajic, A., R. Tauer, H. Feldmann, W. Neupert, and T. Langer. 1994. Yta10p is required for the ATP-dependent degradation of polypeptides in the inner memebrane of mitochondria. FEBS (Fed. Eur. Biochem. Soc.) Lett 353: 201-206 . |
32. | Piskur, J.. 1994. Inheritance of the yeast mitochondrial genome. Plasmid 31: 229-241 |
33. | Simon, V.R., T.C. Swayne, and L.A. Pon. 1995. Actin-dependent mitochondrial motility in mitotic yeast and cell-free systems: identification of a motor activity on the mitochondrial surface. J. Cell Biol 130: 345-354 [Abstract]. |
34. | Smith, M.G., V.R. Simon, H. O'Sullivan, and L.A. Pon. 1995. Organelle-cytoskeletal interactions: actin mutations inhibit meiosis-dependent mitochondrial rearrangement in the budding yeast Saccharomyces cerevisiae. Mol. Biol. Cell 6: 1381-1396 [Abstract]. |
35. | Sogo, L.F., and M.P. Yaffe. 1994. Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane. J. Cell Biol 126: 1361-1373 [Abstract]. |
36. | Strausberg, R.L., and P.S. Perlman. 1978. The effect of zygotic bud position on the transmission of mitochondrial genes in Saccharomyces cerevisiae. Mol. Gen. Genet 163: 131-144 |
37. | Tauer, R., G. Mannhaupt, R. Schanll, A. Pajic, T. Langer, and H. Feldmann. 1994. Yta10p, a member of a novel ATPase family in yeast, is essential for mitochondrial function. FEBS (Fed. Eur. Biochem. Soc.) Lett 353: 197-200 . |
38. | Thomas, D.Y., and D. Wilkie. 1968. Recombination of mitochondrial drug resistance factors in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun 30: 368-372 |
39. | Yaffe, M.. 1991. Organelle inheritance in the yeast cell cycle. Trends Cell Biol 1: 160-164 . |
40. |
Zelenaya-Troitskaya, O.,
S.M. Newman,
K. Okamoto,
P.S. Perlman, and
R.A. Butow.
1998.
Functions of the HMG box protein, Abf2p, in mitochondrial
DNA segregation, recombination and copy number in Saccharomyces cerevisiae.
Genetics
148:
1763-1776
|
41. | Zinn, A.R., J.K. Pohlman, P.S. Perlman, and R.A. Butow. 1987. Kinetic and segregational analysis of mitochondrial DNA recombination in yeast. Plasmid 17: 248-256 |
42. | Zinser, E., and G. Daum. 1995. Isolation and biochemical characterization of organelles from the yeast, Saccharomyces cerevisiae. Yeast 11: 493-536 |