Vivian W.Y. Wong, Amy E. Saunders, Amanda Hutchings, John C. Pascall, Christine Carter, Nicholas A. Bright, Simon A. Walker, Nicholas T. Ktistakis and Geoffrey W. Butcher

Vivian W.Y. Wong

Immunology Program, The Babraham Institute, Cambridge CB22 3AT, UK; Anne McLaren Laboratory for Regenerative Medicine, West Forvie Building, Robinson Way, Addenbrookes Hospital, Cambridge, CB2 0SZ, UK

Amy E. Saunders

Immunology Program, The Babraham Institute, Cambridge CB22 3AT, UK; Department of Microbiology and Immunology, Room 3520, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

Amanda Hutchings

Monoclonal Antibody Facility, The Babraham Institute, Cambridge CB22 3AT, UK

John C. Pascall

Immunology Program, The Babraham Institute, Cambridge CB22 3AT, UK

Christine Carter

Immunology Program, The Babraham Institute, Cambridge CB22 3AT, UK

Nicholas A. Bright

Cambridge Institute for Medical Research, Addenbrooke's Hospital, Cambridge CB2 0XY, UK

Simon A. Walker

Imaging Facility, The Babraham Institute, Cambridge CB22 3AT, UK

Nicholas T. Ktistakis

Signaling Program, The Babraham Institute, Cambridge CB22 3AT, UK

Geoffrey W. Butcher

Corresponding author: geoff.butcher@bbsrc.ac.uk
Immunology Program, The Babraham Institute, Cambridge CB22 3AT, UK

A mutation in the rat GIMAP5 gene predisposes for autoimmunity, most famously in the BB rat model of autoimmune type 1 diabetes mellitus. This mutation is associated with severe peripheral T lymphopenia, as is mutation of the same gene in mice, but the mechanism by which GIMAP5 normally protects T cells from death is unknown. GIMAP5 is a putative small GTPase, a class of proteins which often fulfil their functions in the vicinity of cellular membranes. The objective of this study was to determine the normal intracellular location of GIMAP5 in lymphoid cells. Combining studies in rat, mouse and human systems, novel monoclonal antibodies (mAbs) were used to examine the localisation of GIMAP5, and the closely-related protein, GIMAP1, in lymphoid cells by means of confocal microscopy and sub-cellular fractionation combined with immunoblotting. Additionally, human Jurkat T cells that inducibly express epitope-tagged GIMAP5 were established and used in electron microscopy (EM). Endogenous GIMAP5 was found to be located in a membraneous compartment/s which was also detected by established markers of lysosomes. GIMAP1, by contrast, was found to be located in the Golgi apparatus. EM studies of the inducible Jurkat T cells also found GIMAP5 in lysosomes and, in addition, in multivesicular bodies. This study establishes that the endogenous location of GIMAP5 is in lysosomes and related compartments and provides a clearer context for hypotheses about its mechanism of action.