Are there other components required for Mgm1ps function in mitochondrial structure Destinee Cooper,

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Are there other components required for Mgm1ps
function in mitochondrial structure? Destinee
Cooper, T.J. Holewinske, and Jodi NunnariSection
of Molecular and Cellular Biology, University of
California Davis, Davis, California 95616
Abstract We propose that like Dnm1p, a
dynamin-related GTPase that mediates fission on
the outer membrane, Mgm1p will be regulated by
additional proteins on the inner membrane. We
are conducting a novel enhancer screen to
identify components that assist Mgm1p in its
function to remodel the inner mitochondrial
membrane for efficient mitochondrial fusion.
After mutagenizing mgm1-5 cells with uv rays, we
screened for mutants that enhanced the phenotype
of mgm1-5 cells. In order to ascertain whether
the enhancer mutation was specific to MGM1, we
introduced a copy of wild type MGM1 into
mutagenized cells containing putative enhancer
mutations and tested whether MGM1 restored wild
type growth. Next, the mutant was backcrossed to
mgm1-5 to ensure that only a mutation in a single
locus was conferring the enhanced phenotype. The
mutant was also backcrossed to wild type to
obtain haploid cells containing the enhancer
mutation alone and assess its phenotype. If
enhancer mutants were specific to MGM1, we
proceeded to clone, identify, and characterize.
The primary screen yielded four mutants. After
the secondary screens, one of the mutants was
discarded. The secondary screen analysis on the
second mutant is forthcoming. Third and fourth
mutants are being currently cloned and prepared
for characterization.
Introduction Mitochondria are double membrane
organelles that are the site of aerobic
respiration in eukaryotic cells. Mitochondria
size, shape, and number are dictated by the needs
of the cell. In our model organism,
Saccharomyces cerevisiae (yeast), mitochondria
are localized to the cell cortex and have a
highly branched structure. This structure is
maintained by a balance of fusion and fission
events (3). In yeast cells, components that
mediate membrane fusion and fission have been
characterized, such as Dnm1p, a dynamin-related
GTPase, which mediates fission on the outer
membrane (1). Recently, other proteins, Mdv1p
and Fis1p, have been identified that act together
to regulate Dnm1ps function in fission (2,4).
Fis1p regulates Dnm1ps assembly to the outer
membrane while Fis1p and Mdv1p mediate Dnm1ps
function in mitochondrial membrane constriction
and/or division (4). Another dynamin-related
GTPase, Mgm1p, has been identified that is
postulated to regulate inner membrane dynamics to
promote fusion (5). We propose that like Dnm1p,
Mgm1p will also be regulated by additional
proteins. Weve recently identified two outer
membrane proteins that form a complex with Mgm1p
to mediate fusion (6). Using a genetic approach,
our goal is to identify components that regulate
Mgm1p function.
Results After screening the mutagenized mgm1-5
colonies, four mutants were isolated displaying
an enhanced phenotype of no growth at
temperature(s) that mgm1-5 grows. The mutants
were labeled e(mgm1-5)m6, e(mgm1-5)m11,
e(mgm1-5)m16, and e(mgm1-5)m51. After showing an
enhancement of the mgm1-5 phenotype at a lower
temperature (table 1), the mutants were
transformed with wild type MGM1 (secondary
screen). Based on analysis of secondary screen,
e(mgm1-5)m51 was dropped because it did not show
a recovery of the wild type phenotype (table 2).
At the tertiary screen, the mutants were
backcross to mgm1-5 cells to confirm that the
enhanced phenotype was the result of a single
mutation. E(mgm1-5)m11 and e(mgm1-5)m6 passed
the tertiary screen because they showed a
phenotype ratio of 22 after being backcrossed to
mgm1-5. E(mgm1-5)m6 has been cloned, has five
ORFs (open reading frames), and we are presently
identifying the compliment ORF among them.
E(mgm1-5)m11 has been transformed with a plasmid
library and is currently undergoing screening for
the successful clones. Tertiary screening
results for e(mgm1-5)m16 is pending.
Conclusion We have identified mutations that
enhance MGM1 phenotypes. We have successfully
cloned and are in the process of characterizing
of these, e(mgm1-5)m6. E(mgm1-5)m11 is being
cloned and (E(mgm1-5)m16 is pending further
analysis.) Characterization of the wild type
alleles of the enhancer genes will lead to a
better understanding of Mgm1ps function in inner
membrane remodeling and mitochondrial fusion.
References 1. Bleazard W., McCaffery J.M., King
E.J., Bale S., Mozdy A., Tieu Q., Nunnari J.,
Shaw JM. The dynamin-related GTPases, Dnm1,
regulates mitochondrial fission in yeast. Nat.
Cell Biol. 1298-304 (1999). 2. Mozdy AD,
McCaffery JM, Shaw JM. Dnm1p GTPase-mediated
mitochondrial fission is a multi-step process
requiring the novel integral membrane component
Fis1p. J. Cell Biol. 151367-379 (2000). 3.
Nunnari J., Marshall W., Straight A., Murray A.,
Sedat J.W., Walter P. Mitochondrial transmission
during mating in S. cerevisiae is determined by
mitochondrial fusion and fission and the
intramitochondrial segregation of mt DNA. Mol.
Biol. Cell. 81233-1242 (1997). 4. Tieu Q.,
Nunnari J. Mdv1p is a WD repeat protein that
interacts with the dynamin-related GTPase, Dnm1p,
to trigger mitochondrial division. J. Cell Biol.
151353-365 (2000). 5. Wong E.D., Wagner J.A.,
Gorsich S.W., McCaffery J.M., Shaw J.M., Nunnari
J. The dynamin-related GTPase, Mgm1p, is an
intermembrane space protein required for
maintenance of fusion competent mitochondria. J.
Cell Biol. 151341-352 (2000). 6. Wong E.D.,
Wagner J.A., Scott S.V., Holewinske T.J.,
Cassidy-Stone A., Nunnari J. The
intramitochondrial dynamin-related GTPase, Mgm1p,
is a component of a protein complex that mediates
mitochondrial fusion. Manuscript submitted.
Table 1 Primary screen on YPEG
(ethanol/glycerol) plates. Before 50 kill,
there were about 36154 mutants. Only 69 survived
after replica plating. After streaking for
single colonies, there were 9. Below are the
four that made it past the transformed stage.
Table 2 Secondary screen on YPEG
(ethanol/glycerol) plates. M51 is dropped after
this screen since, at 33 and 34 degrees Celsius,
no recovery of mgm1-5 phenotype is observed.
Materials and Methods A novel enhancer screen
was developed to find single temperature
sensitive mutations that enhance mgm1-5, a
temperature sensitive recessive allele of MGM1.
First, mgm1-5 cells were mutagenized using uv
rays to 50 kill. In the primary screen, mutants
were identified that enhanced the phenotype of
mgm1-5 cells on YPEG, the non-fermentable carbon
source, at 33/34 (semi-permissive temperature).
To establish that enhanced mutant phenotypes were
dependant on the mgm1-5 mutation, we transformed
the mutants with wild type MGM1. To confirm that
the enhanced phenotype was the result of a single
mutation, the mutant was backcross to mgm1-5
cells and haploid colonies were analyzed. If
analysis after the dissecting revealed a 22
ratio of double mutant mgm1-5 and mutant mgm1-5
phenotype, the mutants were cloned introducing a
plasmid library by transformation to the mutant
and observed for complementation. To isolate the
enhancer mutation alone, mutants were backcrossed
to wild type (w303), sporulated, and dissected.
The plasmid is then recovered, amplified in E.
coli, and recovered again. The mutant is then
ready for identification by sequencing and
characterization.
Slow (s) Grows slower than wild type Very slow
(vs) Growth looks sprinkled Wild type
growth /- Some growth, no colonies - No growth
Thank you to the UC LEADS program at UC Davis,
BUSP-SHR, Howard Hughes Medical Institute, the
National Institutes of Health - Initiative for
Minority Student Development , and Hewlett
Packard.