Intracellular localization of the proteasome in response to stress conditions

The ubiquitin-proteasome-system (UPS) fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adaptor proteins Sts1 and Blm10, while in mammalian cells, post-mitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adaptor protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, i.e. quiescence, are associated with a decline in ATP and the re-organization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in a ubiquitin-dependent manner into motile and reversible proteasome storage granules (PSGs) in the cytoplasm. In cancer cells upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing poly-ubiquitinated substrates are formed by liquid-liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling and the intracellular organization of proteasomes during cellular stress conditions

Lipid and protein content profiling of isolated native autophagic vesicles

Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses

A selective autophagy pathway for phase separated endocytic protein deposits. Wilfling et al.

Raw data of westernblot gels, Coomassie stained gels, and microscopy image

Bypassing the nuclear gate: A non-canonical entry pathway for the mitochondrial pyruvate dehydrogenase complex

Zervopoulos et al. (2022) propose a non-canonical nuclear import pathway for the functional mitochondrial pyruvate dehydrogenase complex (PDC), facilitated by dynamic MFN2-mediated tethering of mitochondria to the nuclear envelope upon exposure to proliferative stimuli

A selective autophagy pathway for phase separated endocytic protein deposits. Wilfling et al.

Raw data of westernblot gels, Coomassie stained gels, and microscopy image

Autophagy ENDing unproductive phase-separated endocytic protein deposits

Selective disposal of a wide range of cellular entities by macroautophagy/autophagy is achieved through a special class of proteins called autophagy receptors, which link corresponding cargo to the membrane-bound autophagosomal protein Atg8/LC3. In pursuit of novel autophagy receptors and their cargo, we uncovered a previously undescribed autophagy pathway for removal of aberrant clathrin-mediated endocytosis (CME) protein condensates in S. cerevisiae. Of these CME proteins, Ede1 functions as an autophagy receptor, harboring distinct Atg8-binding domains and driving phase separation into condensates. The aberrant CME condensates at the plasma membrane (PM) exhibit a drop-like structure surrounded by a fenestrated ER, which are engulfed in pieces in an Ede1-dependent manner by autophagy. Thus, our work suggests that aberrant CME is a target for autophagic degradation, with the scaffold protein Ede1 serving as a built-in autophagy receptor that monitors the assembly status of the CME machinery