Starr Matthew L, Hurst Logan R, Fratti Rutilio A
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Traffic. 2016 Oct;17(10):1091-109. doi: 10.1111/tra.12423. Epub 2016 Jul 24.
Yeast vacuole fusion requires the activation of cis-SNARE complexes through priming carried out by Sec18p/N-ethylmaleimide sensitive factor and Sec17p/α-SNAP. The association of Sec18p with vacuolar cis-SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane-associated Sec18p to SNAREs. Thus, we hypothesized that Sec18p associates with PA-rich membrane microdomains before transferring to cis-SNARE complexes upon PA phosphatase activity. Here, we examined the direct binding of Sec18p to liposomes containing PA or DAG. We found that Sec18p preferentially bound to liposomes containing PA compared with those containing DAG by approximately fivefold. Additionally, using a specific PA-binding domain blocked Sec18p binding to PA-liposomes and displaced endogenous Sec18p from isolated vacuoles. Moreover, the direct addition of excess PA blocked the priming activity of isolated vacuoles in a manner similar to chemically inhibiting PA phosphatase activity. These data suggest that the conversion of PA to DAG facilitates the recruitment of Sec18p to cis-SNAREs. Purified vacuoles from yeast lacking the PA phosphatase Pah1p showed reduced Sec18p association with cis-SNAREs and complementation with plasmid-encoded PAH1 or recombinant Pah1p restored the interaction. Taken together, this demonstrates that regulating PA concentrations by Pah1p activity controls SNARE priming by Sec18p.
酵母液泡融合需要通过由Sec18p/N-乙基马来酰亚胺敏感因子和Sec17p/α-SNAP进行的引发来激活顺式SNARE复合体。Sec18p与液泡顺式SNARE的结合部分受磷脂酸（PA）磷酸酶产生二酰基甘油（DAG）的调节。抑制PA磷酸酶活性会阻止膜相关的Sec18p向SNARE的转移。因此，我们推测Sec18p在PA磷酸酶活性作用下转移到顺式SNARE复合体之前，先与富含PA的膜微结构域结合。在此，我们检测了Sec18p与含有PA或DAG的脂质体的直接结合。我们发现，与含有DAG的脂质体相比，Sec18p优先与含有PA的脂质体结合，约为五倍。此外，使用特定的PA结合结构域可阻断Sec18p与PA脂质体的结合，并将内源性Sec18p从分离的液泡中置换出来。而且，直接添加过量的PA会以类似于化学抑制PA磷酸酶活性的方式阻断分离液泡的引发活性。这些数据表明，PA向DAG的转化促进了Sec18p向顺式SNARE的募集。从缺乏PA磷酸酶Pah1p的酵母中纯化的液泡显示，Sec18p与顺式SNARE的结合减少，用质粒编码的PAH1或重组Pah1p进行互补可恢复这种相互作用。综上所述，这表明通过Pah1p活性调节PA浓度可控制Sec18p介导的SNARE引发。
