Di-arginine and FFAT-like motifs retain a subpopulation of PRA1 at ER-mitochondria membrane contact sites.

Prenylated Rab Acceptor 1 (PRA1/Rabac1) is a four-pass transmembrane protein that has been found to localize to the Golgi and promiscuously associate with a diverse array of Rab GTPases. We have previously identified PRA1 to be among the earliest significantly down-regulated genes in the rd1 mouse model of retinitis pigmentosa, a retinal degenerative disease. Here, we show that an endogenous subpopulation of PRA1 resides within the endoplasmic reticulum (ER) at ER-mitochondria membrane contact sites in cultured mammalian cells. We also demonstrate that PRA1 contains two previously unidentified ER retention/retrieval amino acid sequences on its cytosolic N-terminal region: a membrane distal di-arginine motif and a novel membrane proximal FFAT-like motif. Using a truncation construct that lacks complete Golgi targeting information, we show that mutation of either motif leads to an increase in cell surface localization, while mutation of both motifs exhibits an additive effect. We also present evidence that illustrates that N- or C- terminal addition of a tag to full-length PRA1 leads to differential localization to either the Golgi or reticular ER, phenotypes that do not completely mirror endogenous protein localization. The presence of multiple ER retention motifs on the PRA1 N-terminal region further suggests that it has a functional role within the ER.