Home Institution
Ruprecht-Karls-Universität Heidelberg

Host Institution
Gladstone Institute of Virology and Immunology, University of California San Francisco
Mentor: Eric Verdin, Senior Investigator, Professor of Medicine

E-Mail
b.schwer@dkfz-heidelberg.de

Helpful Hints for Future Students
- Get a Safeway Club Card.
- Do not get a bicycle.
- Check out www.craigslist.org and then finding a room, a car, TV, a parrot, etc. will not end up under "greatest difficulties encountered".
- Try not to find a room before you arrive - there is always a nice person around who will accommodate you for the first days.
- Read Thomas Pynchon's "The Crying of Lot 49"

In Saccharomyces cerevisiae, the Sir2 (Silent Information Regulator) protein is associated with chromatin silencing and has also been found to influence cellular metabolism and aging. In Caenorhabditis elegans an increased dosage of a Sir-2 gene extends lifespan by up to 50% percent (Tissenbaum, Nature 410, 227-230; 2001). Furthermore, a novel NAD (nicotinamide adenine dinucleotide)-dependent histone deacetylase activity was recently demonstrated for both yeast and mouse Sir2 (Imai, Nature 403, 795-800; 2000).

Human Sir2 homologues have also been isolated. I worked on the characterization of one of these homologues - hSIRT3 - and its mitochondrial localization. So far, hSIRT3 is the only mitochondrial Sir2 protein. Through import experiments using radioactive in vitro translated protein and isolated mitochondria, I was able to define the requirements for mitochondrial import of hSIRT3. It turned out that the import of hSIRT3 was dependent on the mitochondrial transmembrane potential and that the protein was sorted through the outer and inner mitochondrial membrane into the mitochondrial matrix, where the mitochondrial DNA resides. Mutation of amino acid residues within hSIRT3 showed that mitochondrial targeting was dependent on a stretch of 25 amino acids within the NH2-terminal domain of the protein. Deletion of this region abrogated mitochondrial import of the human Sir2-homologue. Mutation of single amino acids showed that a stretch of positively charged arginine residues within the NH2-terminus was critical for the mitochondrial import of hSIRT3. According to protein structure prediction, this region most likely forms an amphiphilic alpha helix, a structure that is often found within the targeting signal of mitochondrial proteins.

Mitochondria isolated from human cells indeed contained NAD-dependent deacetylase activity and among the currently known human Sir2 homologues with NAD-dependent deacetylase activity, only hSIRT3 could contribute to mitochondrial deacetylase activity.

After import into the mitochondrial matrix, processing of hSIRT3 into a smaller form was observed. Following experiments revealed that hSIRT3 was processed by another protein, the mitochondrial matrix processing peptidase. This processing removed the mitochondrial targeting signal from hSIRT3 after the protein had reached its final destination within the mitochondrial matrix. In vitro experiments using recombinant yeast mitochondrial matrix processing peptidase and hSIRT3 protein suggested that hSIRT3 can only act as an NAD-dependent deacetylase after the NH2 terminus responsible for mitochondrial targeting has been removed by proteolytic cleavage. A detailed report of this work has been published:

Schwer, B., North, B. J., Frye, R. A., Ott, M. & Verdin, E. The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase. J. Cell Biol. 158, 647-657 (2002). http://www.jcb.org/cgi/content/abstract/158/4/647

I especially thank my mentors Dr. Melanie Ott and Dr. Eric Verdin for their continuous support and encouragement and Brian J. North for inspiration and insightful discussions on the world of Sir proteins.