Bue Catherine A, Barlowe Charles
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Biol Chem. 2009 Sep 4;284(36):24049-60. doi: 10.1074/jbc.M109.022590. Epub 2009 Jul 1.
Efficient export of secretory alkaline phosphatase (ALP) from the endoplasmic reticulum depends on the conserved transmembrane sorting adaptor Erv26p/Svp26p. In the present study we investigated the mechanism by which Erv26p couples pro-ALP to the coat protein complex II (COPII) export machinery. Site-specific mutations were introduced into Erv26p, and mutant proteins were assessed in cell-free assays that monitor interactions with pro-ALP cargo and packaging into COPII vesicles. Mutations in the second and third loop domains of Erv26p inhibited interaction with pro-ALP, whereas mutations in the C-terminal tail sequence influenced incorporation into COPII vesicles and subcellular distribution. Interestingly mutations in the second loop domain also influenced Erv26p homodimer associations. Finally we demonstrated that Ktr3p, a cis-Golgi-localized mannosyltransferase, also relies on Erv26p for efficient COPII-dependent export from the endoplasmic reticulum. These findings demonstrate that Erv26p acts as a protein sorting adaptor for a variety of Type II transmembrane cargo proteins and requires domain-specific interactions with both cargo and coat subunits to promote efficient secretory protein transport.
分泌性碱性磷酸酶（ALP）从内质网的高效输出依赖于保守的跨膜分选衔接蛋白Erv26p/Svp26p。在本研究中，我们探究了Erv26p将前体ALP与II型被膜蛋白复合物（COPII）输出机制偶联的机制。将位点特异性突变引入Erv26p，并在无细胞测定中评估突变蛋白，该测定监测与前体ALP货物的相互作用以及包装到COPII囊泡中的情况。Erv26p的第二个和第三个环结构域中的突变抑制了与前体ALP的相互作用，而C末端尾部序列中的突变影响了COPII囊泡的掺入和亚细胞分布。有趣的是，第二个环结构域中的突变也影响了Erv26p同二聚体的缔合。最后，我们证明了顺式高尔基体定位的甘露糖基转移酶Ktr3p也依赖于Erv26p从内质网进行高效的COPII依赖性输出。这些发现表明，Erv26p作为多种II型跨膜货物蛋白的蛋白质分选衔接蛋白，并且需要与货物和被膜亚基进行结构域特异性相互作用以促进高效的分泌蛋白运输。
