(496.5) Proteasome Shuttle Factors Regulate the Relocalization of Proteasomes to Cytosolic Granules upon Specific Stress Conditions

Poster Board Number: A171

Jeroen Roelofs (University of Kansas Medical Center), Gabrielle Vontz (University of Kansas Medical Center, University of Kansas Medical Center), Kenrick Waite (University of Kansas Medical Center), Stella Lee (University of Kansas Medical Center)

Proteasomes are essential complexes within cells responsible for the degradation of many short-lived and aberrant proteins. These substrates are marked for degradation by post-translational modifications with ubiquitin.  This ubiquitination enables substrate recognition by three receptors that are intrinsic subunits of the proteasome. Alternatively, shuttle factors (Rad23, Dsk2, Ddi1 in yeast; a family of orthologs in humans) act as extrinsic receptors and can deliver ubiquitinated substrates to proteasomes. As the human orthologues of Dsk2, Ubiquilin 1 and 2, have been shown to contribute to autophagic processes, we hypothesized that the shuttle factors contribute to the autophagy of proteasomes in yeast. In proliferating cells, proteasomes are largely nuclear; however, upon nitrogen starvation proteasomes exit the nucleus and are degraded in the vacuole through an autophagy pathway known as proteaphagy.  While our biochemical and microscopic analyses indicated that shuttle factors are not required for proteaphagy induced by either nitrogen starvation or proteasome inhibition, we discovered an unexpected role for the shuttle factors in regulating proteasome localization under other stress conditions. Yeast grown to stationary phase or exposed to acute glucose starvation respond by sequestering proteasomes into cytoplasmic granules termed proteasome storage granules (PSGs). These granules behave like liquid-liquid phase separated structures and they appear to protect proteasomes from autophagic degradation. We recently identified a new condition that induces PSG formation, namely mitochondrial inhibition (e.g. using sodium azide). Rad23 and Dsk2 co-localized with PSGs and were important for their formation upon mitochondrial inhibition. Under these conditions, deletion of both genes almost completely prevented granule formation, while single knockouts displayed a partial defect. Surprisingly, neither protein is required for proteasome re-localization to PSGs upon abrupt glucose starvation. Further distinguishing these shuttle factor-dependent granules is their dependance on active protein translation, as cycloheximide treatment prevented their formation, while having no effect on other granule inducing conditions. Our data show granules with unique properties and requirements form depending on the cellular conditions. Finally, we show that the third shuttle factor, Ddi1, has an opposing role in regulating PSGs. The deletion of Ddi1 induced proteasome localization to granules strikingly more quickly compared to wild type cells. We propose a model where the shuttle factors Dsk2 and Rad23 play a key role in facilitating the liquid-liquid phase separation of proteasomes.

NIH-NIGMS R01GM118660 to JR; NIH-NIGMS K-INBRE program P20GM103418 to J.R.