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© The Rockefeller University Press, 0021-9525/1997//287 $5.00
The Journal of Cell Biology, Volume 136, Number 2, , 1997 287-297

Article

Two Separate Signals Act Independently to Localize a Yeast Late Golgi Membrane Protein through a Combination of Retrieval and Retention



Nia J. Bryant and Tom H. Stevens

Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229

The localization of proteins to late-Golgi membranes (TGN) of Saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. These signals could act by directing retrieval from a post-Golgi compartment or by preventing exit from the TGN. To investigate the mechanism of localization of yeast TGN proteins, we used the heterologous protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [ALP]), which localizes to the yeast TGN. Insertion of the aromatic residue–based TGN localization motif (FXFXD) of DPAP A into the cytosolic domain of ALP results in a protein that resides in the TGN. We demonstrate that the FXFXD motif confers Golgi localization through retrieval from a post-Golgi compartment by detecting a post-Golgi processed form of this protein in the TGN. We present an assay that uncouples retrieval-mediated Golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast TGN. We also demonstrate that the cytosolic domain of DPAP A contains additional information, separate from the retrieval motif, that slows exit from the TGN. We propose a model for DPAP A localization that involves two distinct mechanisms: one in which the FXFXD motif directs retrieval from a post-Golgi compartment, and a second that slows the rate at which DPAP A exits the TGN.

Abbreviations used in this paper: ALP, alkaline phosphatase; CPY, carboxypeptidase Y; DPAP A, dipeptidyl aminopeptidase A; E, extracellular; I, intracellular; proCPY, CPY precursor; PM, plasma membrane; PVC, prevacuolar compartment; RS-ALP, retention sequence ALP.

Address all correspondence to Tom H. Stevens, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229. Tel.: (541) 3465884. Fax: (541) 346-4854. e-mail: stevens{at}molbio.uoregon.edu


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