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 DNA-encoded 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 MUTATIONSMATERNAL INHERITANCE OF mtDNA MUTATIONS

http://www.mdausa.org/publications/Quest/q64mito2.htmlhttp://www.mdausa.org/publications/Quest/q64mito2.html

E. Schon, Personal Communication

Some Disease Caused by Mitochondrial Damage

Pearson Large scale deletionsSyndrome

NARP 8993T>G

MIDD 3243A>G

CPEO 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

• 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

• 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

From Zullo, et al 2005

The gene product allowed the cells to grow in the presence of oligomycin proving transformation took place

transformed cells

- oligomycin

+ oligomycin

Sensitive cells

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 productInto 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

ALDHND2

ATPase6

Values above the line are for hydrophobic amino acid, below for hydrophilic ones

(Nuclear coded)

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 productInto a mitochondrial inner membrane

Oxa1 Complex

Oxa1p is nuclear coded and comes intoMatrix 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