2 SEPARATE SIGNALS ACT INDEPENDENTLY TO LOCALIZE A YEAST LATE GOLGI MEMBRANE-PROTEIN THROUGH A COMBINATION OF RETRIEVAL AND RETENTION

The localization of proteins to late-Golgi membranes (TGN) of Saccharo myces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. These signals cou ld act by directing retrieval from a post-Golgi compartment or by prev enting exit from the TGN. To investigate the mechanism of localization of yeast TGN proteins, we used the heterologous protein A-ALP (consis ting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] f used to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [ALP]), which localizes to the yeast TGN. Inserti on of the aromatic residue-based TGN localization motif (FXFXD) of DPA P A into the cytosolic domain of ALP results in a protein that resides in the TGN. We demonstrate that the FXFXD motif confers Golgi localiz ation through retrieval from a post-Golgi compartment by detecting a p ost-Golgi processed form of this protein in the TGN. We present an ass ay that uncouples retrieval-mediated Golgi localization from static re tention-based localization, allowing measurement of the rate at which proteins exit the yeast TGN. We also demonstrate that the cytosolic do main of DPAP A contains additional information, separate from the retr ieval motif, that slows exit from the TGN. We propose a model for DPAP A localization that involves two distinct mechanisms: one in which th e FXFXD motif directs retrieval from a post-Golgi compartment, and a s econd that slows the rate at which DPAP A exits the TGN.