Regulation of proteasome granule formation by proteasome shuttle factors

Abstract

Protein degradation is crucial for many cellular processes including cell cycle regulation, metabolism, and proteostasis. Proteasomes, essential complexes within cells, degrade short-lived and aberrant proteins that are marked for degradation by ubiquitin chains. Although the proteasome houses three intrinsic ubiquitin receptors, other factors, known as shuttle factors, serve as extrinsic ubiquitin receptors that bind ubiquitinated substrates and deliver them to the proteasome for degradation. In yeast, three shuttle factors exist: Rad23, Dsk2, and Ddi1. These factors play a role in a variety of cellular processes including cell cycle regulation, spindle pole body duplication, and DNA damage response. Additionally, a recent study showed ~90% of substrates are transported to proteasomes by Rad23 or Dsk2 indicating shuttle factors are important contributors to ubiquitin-proteasome system dynamics. While generally considered substrate transporters, it is unknown whether shuttle factors influence proteasome localization. In proliferating cells, proteasomes are largely nuclear; however, in response to certain starvation conditions, proteasomes exit the nucleus and are either degraded in the vacuole through an autophagy pathway (proteaphagy) or sequestered into cytoplasmic granules termed proteasome storage granules (PSGs). Under nitrogen starvation, amino acids become limiting and can be supplemented by the degradation of proteasomes. However, the biological advantage of proteasome sequestration into granules in response to carbon starvation is still unknown. In the mammalian system, shuttle factor homologs are involved in autophagy pathways and form liquid droplets reminiscent of PSGs. Here, in yeast, we test for shuttle factor involvement in proteaphagy and PSG formation using fluorescence microscopy. We show shuttle factors are not important for proteaphagy induced by either nitrogen starvation or proteasome inhibition. However, our data reveal that Rad23 and Dsk2 are important for proteasome localization to granules under certain conditions. Specifically, when carbon is gradually depleted upon cell proliferation or ATP production is blocked by sodium azide treatment, the deletion of RAD23 or DSK2 reduces the efficiency of proteasome granule formation. Under these conditions, deletion of both proteins almost completely prevents granules. Interestingly, PSGs induced by abrupt carbon starvation do not depend on Rad23 or Dsk2. This qualitative difference between these granules is corroborated by our observation that active protein translation is required for granule formation under gradual carbon starvation or sodium azide treatment, but not essential for PSGs induced by abrupt starvation. Finally, we show Ddi1, in direct opposition to Rad23 and Dsk2, prevents premature formation of proteasome granules. Cells deleted for DDI1 exhibit granules even in the absence of inducing conditions. In sum, we describe a role for Rad23 and Dsk2 in proteasome localization to granules under certain conditions and distinguish Ddi1 as a proteasome granule inhibitor.