Investigation of Ldb19/Art1 localization and function at the late Golgi

The arrestin-related family of proteins (ARTs) are potent regulators of membrane traffic at multiple cellular locations in the yeast Saccharomyces cerevisiae. Several ARTs act at multiple locations, suggesting that ARTs with well-established functions at one location may have additional, as of yet, uncharacterized roles at other locations in the cell. To more fully understand the spectrum of cellular functions regulated by ART proteins, we explored the localization and function of Ldb19/Art1, which has previously been shown to function at the plasma membrane, yet is reported to localize to the trans-Golgi network (TGN). We report that the C-terminal fusion of Ldb19 with GFP is functional and, as previously reported, localizes to the TGN. We further establish that Ldb19 associates with late stages of TGN maturation that are enriched in the clathrin adaptor protein complex-1 (AP-1). Additionally, we present genetic interaction assays that suggest Ldb19 acts at the late TGN in a mechanism related to that of AP-1. However, Ldb19 and AP-1 have dissimilar phenotypes in a subset of assays of membrane traffic, suggesting Ldb19 functions at the TGN are distinct from those of AP-1. Together these results indicate Ldb19 functions at the TGN, in addition to its well-established role in endocytosis.

抑制蛋白相关蛋白家族（Arrestin-related family of proteins, ARTs）是酿酒酵母（Saccharomyces cerevisiae）中多种细胞位点处膜运输的强效调控因子。部分ARTs可作用于多个位点，这提示在某一位点功能已明确的ARTs，可能在细胞内其他位点存在尚未被阐释的额外功能。为更全面地解析ART蛋白所调控的细胞功能谱，我们对Ldb19/Art1的定位与功能展开了研究：此前已有研究表明该蛋白可定位于质膜，同时也有报道称其定位于反式高尔基网络（trans-Golgi Network, TGN）。本研究证实，Ldb19与绿色荧光蛋白（Green Fluorescent Protein, GFP）的C端融合蛋白具有正常功能，且如此前报道所示，可定位于TGN。我们进一步明确，Ldb19可与富集网格蛋白衔接蛋白复合物1（Clathrin Adaptor Protein Complex-1, AP-1）的TGN成熟晚期阶段相关联。此外，我们通过遗传相互作用实验证实，Ldb19在晚期TGN发挥作用的机制与AP-1相关。然而，在部分膜运输相关实验中，Ldb19与AP-1的表型存在差异，这表明Ldb19在TGN的功能与AP-1并不完全相同。综合以上结果，我们认为Ldb19除了其已被广泛证实的内吞作用功能外，还可在TGN发挥调控功能。