Membrane topography and topogenesis of prenylated rab acceptor (PRA1)

The mouse prenylated Rab acceptor (mPRA1) is associated with the Golgi memb rane at steady state and interacts with Rab proteins. It contains two inter nal hydrophobic domains (34 residues each) that have enough residues to for m four transmembrane (TM) segments. In this study, we have determined the m embrane topography of mPRA1 in both intact cells and isolated microsomes. T he putative TM segments of mPRA1 were used to substitute for a known TM seg ment of a model membrane protein to determine whether the mPRA1 segments in tegrate into the membrane. Furthermore, N-linked glycosylation scanning met hods were used to distinguish luminal domains from cytoplasmic domains of m PRA1. The data demonstrate that mPRA1 is a polytopic membrane protein conta ining four TM segments. These TM segments act cooperatively during the tran slocation and integration at the endoplasmic reticulum membrane. All hydrop hilic domains are in the cytoplasm, including the N-terminal domain, the li nker domain between the two hydrophobic domains, and the C-terminal domain. As a result, the bulk of mPRA1 is located in the cytoplasm, supporting its postulated role in regulating Rab membrane targeting and intracellular tra fficking.