Human and Viral Golgi Anti-apoptotic Proteins (GAAPs) Oligomerize via Different Mechanisms and Monomeric

Golgi anti-apoptotic proteins (GAAPs) are hydrophobic pro- teins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca 2 content of intracellular stores, and regulate Ca 2 fluxes. GAAP was dis- covered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca 2 -modulating membrane proteins. Most TMBIM mem- bers show multiple bands when analyzed by SDS-PAGE, sug- gesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligo- merization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligo- merization of hGAAP is independent of cysteines, but oligomeri- zation of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellu- lar Ca 2 stores. In conclusion, GAAP can oligomerize in a pH-reg-