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New complex mitochondrial functions in cell biology
P a r t i c i p a n t s :
Claudio Falcone, professor; Silvia Francisci, Teresa Rinaldi,
Cristina Mazzoni, researchers; Cristina De Luca, Vanessa
Palermo, post-doc fellows; Michele Saliola, technician.
C o l l a b o r a t i o n s :
Laboratory of Molecular Genetics, Université Paris Sud, Orsay,
France (Prof. Monique Bolotin-Fukuhara); Department of
Biology, The Technion, University of Haifa, Israel (Dr. Michael
Glickman).
Report of activity
Our work was planned to connect three aspects of
mitochondrial functions in cell life, apoptosis and
disease.
The first research line investigates the role of the
proteasome in regulating mitochondrial fusion and
fission events, which are essential in maintaining the
mitochondrial shape. This highly dynamic system is
conserved in evolution and is altered in some human
diseases. We have shown that the proteasomal
mutant (rpn11-m1) exhibits fragmented mitochondria
and a very peculiar cell cycle defect. Since the
mpr1 protein lacks the last 31 aminoacids of the wt
Rpn11 protein, which is the deubiquitinating
enzyme of the proteasome, we investigated in detail
the function of these aminoacids. As previously suggested
by the genetic analysis of the rpn11 extragenic
revertants, the cell cycle and mitochondrial
defects can be separated, so we have performed a site
specific mutagenesis in order to identify the
aminoacids involved in maintaining the correct
mitochondrial morphology and those necessary for
the correct cell cycle. We have identified a putative
α-helix necessary for the maintenance of the correct
cell cycle, while a very short region of the C terminal
part of Rpn11 was found to be essential for the
maintenance of the tubular mitochondrial morphology.
Furthermore we have shown that expression of
Principal investigator: Laura Frontali
Professor of Microbial Chemistry
Dipartimento di Biologia Cellulare e dello Sviluppo
Tel: (+39) 06 4453950, Fax: (+39) 06 4461980
laura.frontali@uniroma1.it
49
Molecular genetics of eukaryotes - AREA 3
the C-terminal part of Rpn11 is able to complement
in trans all the pleiotropic phenotypes of the rpn11m1
mutant; this result suggests that the Rpn11 protein
could act in controlling the mitochondrial shape
even independently from the proteasome. Finally we
have investigated the mechanisms by which Rpn11
controls the mitochondrial shape and show that
Rpn11 may regulate the mitochondrial fission and
tubulation processes.
In the second line of research, we have concentrated
our analysis on aging and apoptosis. Mitochondrial
morphology changes from a network shape to a
punctuate one during aging and following an apoptotic
stimulus. The significance of these alterations
is not known, but it is clear that an excess of fission
events in mitochondria and/or a defective mitochondrial
fusion results in destruction of the mitochondrial
tubular network with consequent respiratory
defect, accumulation of ROS, and apoptosis in mammalian
cells. We found that the deletion of the
DNM1, known to be protective from apoptotic triggers,
leads also to extended longevity. The F-box
MDM30/DSG1 is known to affect mitochondrial
morphology, but its role in aging and apoptosis has
never been investigated. We showed that the mdm30
null mutant presents extended longevity and high
resistance to oxidative stress, suggesting a role for
this gene in both apoptosis and aging. Yme1p is a
subunit, together with Mgr1p, of the mitochondrial
inner membrane i-AAA protease complex, which is
responsible for degradation of unfolded or misfolded
mitochondrial proteins. Yme1p shows 42% and 33%
homology with the human proteins YME1L and
paraplegin (SPG7), respectively. We demonstrated
that the deletion of YME1 leads to premature loss of
viability during chronological aging and to increased
sensitivity to apoptotic stimuli.
Lastly we have confirmed the possibility of using
yeast mitochondria as a flexible and versatile tool to