Bryant N J, Stevens T H
Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.
J Cell Biol. 1997 Jan 27;136(2):287-97. doi: 10.1083/jcb.136.2.287.
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.
酿酒酵母中蛋白质定位于晚期高尔基体膜（反式高尔基体网络，TGN）是由这些蛋白质胞质结构域中含芳香族残基的靶向基序赋予的。这些信号可能通过指导从高尔基体后区室的回收作用，或通过阻止从TGN输出而起作用。为了研究酵母TGN蛋白的定位机制，我们使用了异源蛋白A-碱性磷酸酶（由二肽基氨基肽酶A [DPAP A]的胞质结构域与液泡蛋白碱性磷酸酶[ALP]的跨膜和腔结构域融合而成），它定位于酵母TGN。将基于芳香族残基的DPAP A的TGN定位基序（FXFXD）插入ALP的胞质结构域，会产生一种定位于TGN的蛋白。我们通过在TGN中检测到这种蛋白的高尔基体后加工形式，证明FXFXD基序通过从高尔基体后区室的回收作用赋予高尔基体定位。我们提出了一种检测方法，可将回收介导的高尔基体定位与基于静态保留的定位解偶联，从而能够测量蛋白质从酵母TGN输出的速率。我们还证明，DPAP A的胞质结构域包含与回收基序不同的额外信息，该信息会减缓从TGN的输出。我们提出了一个DPAP A定位模型，该模型涉及两种不同机制：一种是FXFXD基序指导从高尔基体后区室的回收作用，另一种是减缓DPAP A从TGN输出的速率。
