Mukherjee Indrani, Barlowe Charles
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755.
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755
Mol Biol Cell. 2016 May 15;27(10):1635-49. doi: 10.1091/mbc.E15-10-0704. Epub 2016 Mar 30.
SLY41 was identified as a multicopy suppressor of loss of Ypt1, a Rab GTPase essential for COPII vesicle tethering at the Golgi complex. SLY41 encodes a polytopic membrane protein with homology to a class of solute transporter proteins, but how overexpression suppresses vesicle-tethering deficiencies is not known. Here we show that Sly41 is efficiently packaged into COPII vesicles and actively cycles between the ER and Golgi compartments. SLY41 displays synthetic negative genetic interactions with PMR1, which encodes the major Golgi-localized Ca(2+)/Mn(2+) transporter and suggests that Sly41 influences cellular Ca(2+) and Mn(2+) homeostasis. Experiments using the calcium probe aequorin to measure intracellular Ca(2+) concentrations in live cells reveal that Sly41 overexpression significantly increases cytosolic calcium levels. Although specific substrates of the Sly41 transporter were not identified, our findings indicate that localized overexpression of Sly41 to the early secretory pathway elevates cytosolic calcium levels to suppress vesicle-tethering mutants. In vitro SNARE cross-linking assays were used to directly monitor the influence of Ca(2+) on tethering and fusion of COPII vesicles with Golgi membranes. Strikingly, calcium at suppressive concentrations stimulated SNARE-dependent membrane fusion when vesicle-tethering activity was reduced. These results show that calcium positively regulates the SNARE-dependent fusion stage of ER-Golgi transport.
SLY41被鉴定为Ypt1缺失的多拷贝抑制因子，Ypt1是一种Rab GTP酶，对COPII囊泡在高尔基体复合体处的拴系至关重要。SLY41编码一种与一类溶质转运蛋白具有同源性的多跨膜蛋白，但过表达如何抑制囊泡拴系缺陷尚不清楚。在这里，我们表明Sly41被有效地包装到COPII囊泡中，并在内质网和高尔基体区室之间活跃循环。SLY41与PMR1表现出合成负遗传相互作用，PMR1编码主要定位于高尔基体的Ca(2+)/Mn(2+)转运蛋白，这表明Sly41影响细胞内Ca(2+)和Mn(2+)稳态。使用钙探针水母发光蛋白测量活细胞内Ca(2+)浓度的实验表明，Sly41过表达显著提高了胞质钙水平。尽管未鉴定出Sly41转运蛋白的特定底物，但我们的研究结果表明，Sly41在内质网早期分泌途径中的局部过表达可提高胞质钙水平，以抑制囊泡拴系突变体。体外SNARE交联试验用于直接监测Ca(2+)对COPII囊泡与高尔基体膜拴系和融合的影响。令人惊讶的是，当囊泡拴系活性降低时，抑制浓度的钙刺激了SNARE依赖的膜融合。这些结果表明，钙正向调节内质网-高尔基体运输中SNARE依赖的融合阶段。
