Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Biol Cell. 2020 Nov;112(11):349-367. doi: 10.1111/boc.202000058. Epub 2020 Sep 3.
In the yeast Saccharomyces cerevisiae, acute glucose starvation induces rapid endocytosis followed by vacuolar degradation of many plasma membrane proteins. This process is essential for cell viability, but the regulatory mechanisms that control it remain poorly understood. Under normal growth conditions, a major regulatory decision for endocytic cargo occurs at the trans-Golgi network (TGN) where proteins can recycle back to the plasma membrane or can be recognized by TGN-localised clathrin adaptors that direct them towards the vacuole. However, glucose starvation reduces recycling and alters the localization and post-translational modification of TGN-localised clathrin adaptors. This raises the possibility that during glucose starvation endocytosed proteins are routed to the vacuole by a novel mechanism that bypasses the TGN or does not require TGN-localised clathrin adaptors.
Here, we investigate the role of TGN-localised clathrin adaptors in the traffic of several amino acid permeases, including Can1, during glucose starvation. We find that Can1 transits through the TGN after endocytosis in both starved and normal conditions. Can1 and other amino acid permeases require TGN-localised clathrin adaptors for maximal delivery to the vacuole. Furthermore, these permeases are actively sorted to the vacuole, because ectopically forced de-ubiquitination at the TGN results in the recycling of the Tat1 permase in starved cells. Finally, we report that the Mup1 permease requires the clathrin adaptor Gga2 for vacuolar delivery. In contrast, the clathrin adaptor protein complex AP-1 plays a minor role, potentially in retaining permeases in the TGN, but it is otherwise dispensable for vacuolar delivery.
This work elucidates one membrane trafficking pathway needed for yeast to respond to acute glucose starvation. It also reveals the functions of TGNlocalised clathrin adaptors in this process. Our results indicate that the same machinery is needed for vacuolar protein sorting at the GN in glucose starved cells as is needed in the presence of glucose. In addition, our findings provide further support for the model that the TGN is a transit point for many endocytosed proteins, and that Gga2 and AP-1 function in distinct pathways at the TGN.
在酵母酿酒酵母中，急性葡萄糖饥饿会诱导快速内吞作用，随后液泡降解许多质膜蛋白。这个过程对细胞存活至关重要，但控制它的调节机制仍知之甚少。在正常生长条件下，内吞货物的主要调节决策发生在跨高尔基网络 (TGN)，在那里蛋白质可以回收回质膜，或者可以被 TGN 定位的网格蛋白衔接蛋白识别，将它们导向液泡。然而，葡萄糖饥饿会减少回收，并改变 TGN 定位的网格蛋白衔接蛋白的定位和翻译后修饰。这就提出了这样一种可能性，即在葡萄糖饥饿期间，内吞的蛋白质可能通过一种绕过 TGN 或不需要 TGN 定位的网格蛋白衔接蛋白的新机制被运送到液泡。
在这里，我们研究了 TGN 定位的网格蛋白衔接蛋白在几种氨基酸通透酶（包括 Can1）在葡萄糖饥饿期间的内吞作用中的作用。我们发现，Can1 在饥饿和正常条件下内吞后穿过 TGN。Can1 和其他氨基酸通透酶需要 TGN 定位的网格蛋白衔接蛋白才能最大程度地递送到液泡。此外，这些通透酶被主动分拣到液泡中，因为 TGN 上异位强制去泛素化导致 Tat1 通透酶在饥饿细胞中回收。最后，我们报告说 Mup1 通透酶需要网格蛋白衔接蛋白 Gga2 才能递送到液泡。相比之下，网格蛋白衔接蛋白复合物 AP-1 只起次要作用，可能在将通透酶保留在 TGN 中起作用，但在液泡递送上则是可有可无的。
这项工作阐明了酵母对急性葡萄糖饥饿做出反应所需的一种膜运输途径。它还揭示了 TGN 定位的网格蛋白衔接蛋白在这个过程中的功能。我们的结果表明，在葡萄糖饥饿的细胞中，用于液泡蛋白分拣的 TGN 所需的机制与在有葡萄糖的情况下相同。此外，我们的研究结果进一步支持了 TGN 是许多内吞蛋白的转运点的模型，并且 Gga2 和 AP-1 在 TGN 中起不同的作用。
