Pinar Mario, Arst Herbert N, Pantazopoulou Areti, Tagua Víctor G, de los Ríos Vivian, Rodríguez-Salarichs Javier, Díaz J Fernando, Peñalva Miguel A
Departments of Cellular and Molecular Biology, and.
Departments of Cellular and Molecular Biology, and Section of Microbiology, Imperial College London, London SW7 2AZ, United Kingdom.
Proc Natl Acad Sci U S A. 2015 Apr 7;112(14):4346-51. doi: 10.1073/pnas.1419168112. Epub 2015 Mar 23.
The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabO(RAB1) resides in the Golgi, RabE(RAB11) localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabE(RAB11), but not RabO(RAB1), immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabE(RAB11) in vivo. hypA1(ts) rapidly shifts RabE(RAB11), but not RabO(RAB1), to the cytosol, consistent with HypA(Trs120) being specifically required for RabE(RAB11) activation. Missense mutations rescuing hypA1(ts) at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabO(RAB1) did not, establishing that the essential role of TRAPPII is facilitating RabE(RAB11) nucleotide exchange. In vitro, TRAPPII purified with HypA(Trs120)-S-tag accelerates nucleotide exchange on RabE(RAB11) and, paradoxically, to a lesser yet substantial extent, on RabO(RAB1). Evidence obtained by exploiting hypA1-mediated destabilization of HypA(Trs120)/HypC(Trs130)/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabO(RAB1), which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypA(Trs120) colocalizes with RabE(RAB11), arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi-to-post-Golgi transition.
寡聚复合物转运蛋白颗粒I（TRAPPI）介导RAB GTP酶RAB1/Ypt1上的核苷酸交换。TRAPPII由TRAPPI加上另外三个亚基Trs120、Trs130和Trs65组成。尚不清楚TRAPPII是否介导RAB1/Ypt1、RAB11/Ypt31上的核苷酸交换，还是两者都介导。在构巢曲霉中，RabO（RAB1）定位于高尔基体，RabE（RAB11）定位于正在向顶端运输的高尔基后胞吐载体，hypA编码Trs120。RabE（RAB11）免疫沉淀复合物中含有Trs120/Trs130/Trs65，但RabO（RAB1）免疫沉淀复合物中没有，这表明TRAPPII在体内与RabE（RAB11）存在特异性关联。hypA1（ts）能迅速将RabE（RAB11）而非RabO（RAB1）转移至胞质溶胶，这与HypA（Trs120）是RabE（RAB11）激活所特需的一致。在42°C时挽救hypA1（ts）的错义突变定位到rabE，影响七个残基。其中六个残基的替换，有四个导致GDP释放加速7至36倍，挽救了与TRAPPII缺陷相关的致死性，而RabO（RAB1）中的等效替换则没有，这表明TRAPPII的基本作用是促进RabE（RAB11）的核苷酸交换。在体外，用HypA（Trs120）-S标签纯化的TRAPPII能加速RabE（RAB11）上的核苷酸交换，而且矛盾的是，在较小但仍很显著的程度上也能加速RabO（RAB1）上的核苷酸交换。利用hypA1介导的HypA（Trs120）/HypC（Trs130）/Trs65组装到TRAPPI核心上的去稳定化获得的证据表明，这些亚基在TRAPP上塑造了第二个RAB结合位点，显然独立于RabO（RAB1）的结合位点，这可以解释TRAPPII在体外对两种RAB的活性。利用构巢曲霉体内显微镜技术，我们发现HypA（Trs120）与RabE（RAB11）共定位，在晚期高尔基体潴泡消散为胞吐载体时到达。因此，TRAPPII标记并可能决定了高尔基体到高尔基后阶段的转变。
