University of Western Ontario - Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Dr. JoaquĆ­n Madrenas

Abstract

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial-resident protein and a member of the highly conserved stomatin and SPFH families, which includes Stomatins, Prohibitins, Flotillins and bacterial HflC/K proteins. Studies on SPFH domain-containing family members have pointed to a role in the organization of membranes into functional domains as the mechanism underlying the control of a wide variety of cellular functions. The Madrenas laboratory originally identified SLP-2 in the detergent-insoluble fraction of human T cells activated through the T cell receptor and found that over-expression of SLP-2 increased T cell activation. Based on these results and on the conservation of function across other SPFH family members, we hypothesized that SLP-2 acts to facilitate mitochondrial and plasma membrane organization in response to T cell activation.

To investigate the role of SLP-2 during T cell activation, we generated stably transfected Jurkat T cells expressing GFP-tagged SLP-2 and also produced recombinant human SLP-2 protein. These tools allowed us to demonstrate a direct interaction of SLP-2 with the mitochondrial lipid cardiolipin and to show an association of SLP-2 with the prohibitin complex. Furthermore, an increased recruitment of prohibitin to mitochondrial membranes upon SLP-2 over-expression was also observed. At the cellular level, cells over-expressing SLP-2 were found to have an increase in mitochondrial biogenesis, increased respiratory chain activity and decreased susceptibility to apoptotic induction. Next, T cell-specific SLP-2 conditional knockout mice were generated and found to have altered T cell mitochondrial membrane organization in the absence of SLP-2. SLP-2-deficient T cells showed significantly lower levels of cardiolipin in detergent-resistant mitochondrial fractions, along with decreased complex I levels and activity. The absence of SLP-2 also led to a decrease in T cell activation, which translated into a decreased rejection rate for non-matched tissue transplants. Finally, for the first time it has been shown that SLP-2 is found in two intracellular pools, one in mitochondria and the other associated with plasma membrane, with the plasma membrane pool surrounding T cell receptors upon T cell activation. This implies a fundamental role for SLP-2 in membrane organization.

Taken together, the results presented here demonstrate a role for SLP-2 in the compartmentalization of mitochondrial and plasma membranes into functional domains. This, in turn, affects multi-domain receptor and multi-protein complex assembly and thus affects such functions as mitochondrial biogenesis, respiratory chain activity, apoptotic induction and T cell activation.

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