Professor
(McGill University)
Howard Hughes Medical Institute International Research Scholar
Canada Research Chair in Cell Biology
Canadian Institutes of Health Research Senior Investigator
Fellow of the Royal Society of Canada

Phone: 780-492-9868
Fax: 780-492-9278
Email: rick.rachubinski@ualberta.ca

Molecular Mechanisms of Peroxisome Biogenesis and Prion Formation

Peroxisomes are ubiquitous organelles that are defined as containing at least one hydrogen peroxide-producing oxidase and catalase to decompose the hydrogen peroxide.  They perform many biochemical functions of lipid metabolism, including the β-oxidation of fatty acids.  Mature peroxisomes are spherical, with diameters between 0.5 and 1.0 micrometer.  Each peroxisome is delimited by a single membrane and contains a fine granular matrix.  Peroxisomes do not contain DNA or an independent protein synthesis machinery.  Accordingly, all peroxisomal proteins are encoded in the nucleus and synthesized on cytosolic polysomes.

Peroxisomes are essential for normal human development and physiology, as shown by the lethality of a group of genetic disorders called the peroxisome biogenesis disorders (PBD).  PBD-afflicted individuals fail to assemble functional peroxisomes.  We are therefore interested in defining the molecular cascade of events involved in peroxisome biogenesis.

Electron micrograph of a cell of the yeast Yarrowia lipolytica. P, peroxisome; M, mitochondrion; N, nucleus; NE, nuclear envelope; CW, cell wall; V, vacuole

We use the yeasts Yarrowia lipolytica and Saccharomyces cerevisiae to study peroxisome biogenesis.  Yeasts are ideal models for studying the PBDs for two reasons.  First, the mechanism of peroxisome assembly has been conserved during evolution from yeasts to humans.  Second, there is a conditional requirement for peroxisomes on different carbon sources that makes yeasts ideal genetic model systems with which to study peroxisome biogenesis and has permitted the generation and identification of a number of peroxisome assembly, or pex, mutants that mimic the human PBDs. Complementation of these yeast pex mutants has played a paramount role in the identification of a large number of PEX genes encoding peroxins required for peroxisome assembly.  These yeast genes have been used to scan human gene databases to identify human PEX gene orthologs.  The PEX genes complementing all thirteen complementation groups of the PBDs have now been identified.

Our current research employs global biological approaches like differential gene microarray analysis and proteomics with classical cell biological techniques such as fluorescence microscopy and subcellular fractionation to identify novel peroxin-encoding genes in S. cerevisiae.  We have also embarked on the identification of peroxisomal proteins involved in peroxisome inheritance.  Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Using live-cell 4-dimensional video microscopy, we have recently characterized the first peroxisomal protein, Inp1p for inheritance of peroxisomes protein 1, directly involved in peroxisome inheritance.

High throughput technologies such as mass spectrometry and gene microarrays and comprehensive databases of global gene expression patterns and global protein localizations are invaluable resources for the modern cell biologist.  We have exploited the availability of these resources in novel and exciting ways to examine a fundamental and essential cellular process - peroxisome biogenesis.

Prions are a special class of protein aggregates that replicate their conformation and spread infectiously.  Prions are the agents that cause bovine spongiform encephalopathy (BSE or mad cow disease) and its human equivalent, variant Creutzfeldt-Jakob disease (vCJD).  The molecular mechanism by which a protein is converted from a non-prion state to a prion state is poorly understood.

S. cerevisiae contains several proteins with prion properties.  One of the yeast prion states, called [PSI+], results from a self-perpetuating aggregate of Sup35 (a translational termination factor) and provides an excellent model system with which to examine prion formation and propagation.  The amino-terminal domain (N) and the middle domain (M) of Sup35 are known to be necessary and sufficient for Sup35 to transition between the soluble, functional state [psi] and the prion state [PSI+].  Transient overexpression of the Sup35-NM domain leads to Sup35 aggregation and the de novo appearance of [PSI+].  We are using overexpression of fluorescently labeled Sup35 together with a S. cerevisiae deletion strain library (containing 4828 deleted genes) to elucidate protein factors involved in prion initiation, expansion and inheritance.

Prions have domains that are rich in the amino acids glutamine (Q) and arginine (R) and often have repetitive regions of sequence.  Using a perl script (to determine high R and Q content), on-line bioinformatics tools (to find repetitive regions), and literature interrogation (to find how others define prion domains), we have queried all proteins possibly encoded by the S. cerevisiae genome.  Using a library of S. cerevisiae strains coding for all expected proteins linked to the fluorescent protein GFP, we are testing 138 identified protein candidates for prion-like attributes, such as the formation of one or more bright foci under microscopic observation, curability with protein denaturants and inheritability.

The identification of proteins that affect prion formation will enhance our understanding of diseases of protein misfolding.  A variety of debilitating diseases including Alzheimer’s, Huntington’s, Parkinson’s, BSE and vCJD are all linked to protein misfolding and aggregation.  Elucidation of the molecular basis for the protein conformational changes occurring in one disease will help to understand the molecular bases of the other disorders.

Selected Publications

Graduate Students

Erin Brown

Jenny Chang

David Lancaster

Fred Mast

Robert Tower

Maninder Virk

Postdoctoral Fellows

Dr. Andrei Fagarasanu

Dr. Barbara Knoblach

Technicians

Hanna Kroliczak

Rick Poirier

Elena Savidov

Dwayne Weber