Transcript Slide 1

Factors that might affect the
Allotopic Replacement of a Damaged
Mitochondrial DNA-encoded Protein
S.J. Zullo J.M. Eisenstadt, C.R. Merril, H. Weiner
Stable Transformation of CHO Cells and
Human NARP Cybrids Confers Oligomycin
Resistance (olir) following Transfer of a
Mitochondrial DNA-encoded olir ATPase 6
Gene to the Nuclear Genome: A Model System
for mtDNA Gene Therapy
S.J. Zullo, W.T. Parks, M. Chloupkova, B. Wei, H. Weiner, W.A.
Fenton, J.M. Eisenstadt and C.R. Merril
Rejuvenation Research. 2005 8,18-28.
Earlier Study
Rescue of a deficiency in ATP synthesis by
transfer of MTATP6, a mitochondrial DNAencoded gene, to the nucleus.
Manfredi G, Fu J, Ojaimi J, Sadlock JE, Kwong
JQ, Guy J, Schon EA
Nature Genetics 30, 394-399 (2002)
(This study used a Flag-epitope to show that
incorporation occurred and transient transfections).
MATERNAL INHERITANCE OF mtDNA MUTATIONS
http://www.mdausa.org/publications/Quest/q64mito2.html
E. Schon, Personal Communication
Some Disease Caused by
Mitochondrial Damage
Pearson
Large scale deletions
Syndrome
NARP
MIDD
CPEO
8993T>G
3243A>G
Large scale deletions
Goals for the talk
• Review mitochondrial coded proteins
• Explain the importance of the Zullo
published paper
• Review protein import
• Discuss some of the potential pit-falls
Typical Mammalian Mitochondrial
Genome
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Codes for a limited number of proteins
All are subunits of large complexes
All are involved in electron transfer
All are membrane bound
All are relatively hydrophobic proteins
Genome is more like a bacteria
Codon usage is slightly different
7 su of C-I
1 of C-III
3 0f C-IV
2 of C-V
Damaged Gene Products
• How to repair a damaged gene?
Change or repair the DNA is not easy
• How to replace the protein?
Express it in the nucleus and have it be
transported into mitochondria
Need to change a few codons, but that is not
a major problem
Zullo Paper
• The basic idea was to place the ATPase 6 gene
in the nucleus behind a leader sequence so the
protein could be expressed in the cytosol then
be imported into mitochondria.
• It worked!
• We used an Oligomycin Resistance marker
and in vitro import to show that Allotopic
replacement occurred. This infers that the
protein was inserted properly into Complex 5
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S.J. Zullo, W.T. Parks, M. Chloupkova, B. Wei, H. Weiner, W.A. Fenton, J.M.
Eisenstadt and C.R. Merril
Rejuvenation Research. 2005 8,18-28
Leader Sequence
• These are typically 20-35 amino acids
found at the N-terminal of the protein.
They have the ability to bring virtually any
protein into mitochondria matrix space.
• They are removed by a protease (MPP) as
the protein is imported into the matrix
space.
• Some proteins have additional signals that
allow them to go into the membrane.
Zullo Study
• ATPase 6 possessed an oligomycin
resistant maker. The fact that the cells
were resistant after transformation shows
that the protein was incorporated correctly
into the channel since that is to it where
the drug binds to the susceptible cells.
The proton channel
ATPase 6
ATPase 9
The gene product allowed the cells to grow in the
presence of oligomycin proving transformation took place
- oligomycin
Sensitive cells
transformed cells
+ oligomycin
From Zullo, et al 2005
Potential Problems for Allotopic
Replacement
• Hydrophobicity and Processing
• Assembly into complexes
• Replacing existing damaged protein in a
preformed complex
Import of a nuclear coded gene product
Into a mitochondrial inner membrane
How is a Nuclear Coded Protein
Imported into Mitochondria?
Post-translational
Dogma says protein is
totally synthesized
then is imported using
TOM and TIM
complexes
Co-translational
Our laboratory and
others show that
import could occur in
a co-translational
manner. That is,
import occurs as
protein is coming off
ribosome
Co- vs. Post-translational import
Co-translation
Post-translation
Ribosome
HSPs
Mitochondria
Hydrophobicity Plots
ND1
ND2
ATPase6
ALDH
(Nuclear coded)
Values above the line are for hydrophobic amino acid, below for hydrophilic ones
Will hydrophobic protein import
properly?
• Could bind to other hydrophobic molecules
• Could aggregate in cytosol if post
translational mechanism functions
• ATPase 6 worked in our study but we do
not know if it was assembled properly in
the membrane to allow for ATP formation
but all indications are that is was as
originally shown by Manfredi et al.
How is the Mitochondrial-coded
Protein Inserted?
• Literature says primarily co-translational
• That is the protein goes into the
membrane as it comes off the
mitochondrial ribosome
• This means it is never totally free in the
matrix space and enters membrane from
the matrix side
• A nuclear coded protein could insert from
the space between the membrane
Import of a nuclear coded gene product
Into a mitochondrial inner membrane
Oxa1 Complex
Oxa1p is nuclear coded and comes into
Matrix then is inserted into membrane
Assembly
• Mitochondrial coded proteins are part of
large complexes
• The assembly of these complexes is not
well understood
• Can you replace a protein already in the
complex?
BC1-Complex
Assembly vs Replacement
• Assembly
This means that the
new protein would
have to assemble
with the other
subunits so it can be
inserted properly into
the membrane.
Scaffold is important
• Replacement
This means the new
protein would have to
replace a protein that
was an integral part of
the complex. To
occur the complex
might have to
dissociate and
reassemble
Mitochondria Biogenesis
• Even if the protein can not replace a
damaged or missing protein it might be
able to be incorporated during
mitochondrial biogenesis.
• Little is known about the assembly of
complexes during cell division.
Summary
• We showed that the ATPase 6 mitochondrial gene
can be placed in the nucleus and the resulting
protein will be imported into the matrix space.
• It will be necessary to do this with the dozen other
mitochondrial genes to know if Allotopic
Replacement is a viable option.
• It will have to be proved that the various proteins
can assemble and function in the complexes.
Last point
We just found that a bacterial signal sequence
can bring a protein into mitochondria. The
physiological significance is not yet know but
this might prove to be component to cellular
damage or an area worth exploring.
Mukhopadhyay, Ni, Yang and Weiner (2005) Bacterial Signal Peptide that
recognizes HeLa cell Mitochondrial Import Receptor components is a
functional Mitochondrial Import Leader Sequence. Cell and Molecular
Life Sciences In press.
Bacterial Leader
• pALDH leader (rat liver)MLRAALSTARRGPRLSRLL
• Toho-1 leader (E. coli)MMTQSIRRSMLTVMATLPLLFSSATLHAQAN
• The bacterial leader does not have properties
found typically in a mitochondrial leader
sequences, yet this one brought proteins into
HeLa cell mitochondria matrix space.
Acknowledgements
• Steve Zullo for involving me with the first
study
• Abhijit Mukhopadhyay at Purdue
• Aubrey and the organizers for inviting me
• You for your attention