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AMPK Regulates ESCRT-Dependent Microautophagy of Proteasomes Concomitant With bioRxiv preprint doi: https://doi.org/10.1101/751420; this version posted August 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 AMPK regulates ESCRT-dependent microautophagy of proteasomes concomitant with 4 proteasome storage granule assembly during glucose starvation 5 6 Short title: AMPK and ESCRT-dependent proteasome microautophagy 7 8 Jianhui Li1, Michal Breker3, Morven Graham4, Maya Schuldiner3, Mark Hochstrasser1,2* 9 10 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 11 Connecticut, United States of America 12 13 2Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, 14 Connecticut, United States of America 15 16 3Department of Molecular Genetics, Weizmann Institute of Sciences, Rehovot, Israel 17 18 4 Center for Cellular and Molecular Imaging, School of Medicine, Yale University, New Haven, 19 Connecticut, United States of America 20 21 22 * Corresponding author 23 E-mail: [email protected] (MH) 24 bioRxiv preprint doi: https://doi.org/10.1101/751420; this version posted August 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 25 Abstract 26 The ubiquitin-proteasome system regulates numerous cellular processes and is central to 27 protein homeostasis. In proliferating yeast and many mammalian cells, proteasomes are highly 28 enriched in the nucleus. In carbon-starved yeast, proteasomes migrate to the cytoplasm and collect 29 in phase-separated proteasome storage granules (PSGs). PSGs dissolve and proteasomes return to 30 the nucleus within minutes of glucose refeeding. The mechanisms by which cells regulate 31 proteasome homeostasis under these conditions remain largely unknown. Here we show that AMP- 32 activated protein kinase (AMPK) together with endosomal sorting complexes required for 33 transport (ESCRTs) drive a glucose starvation-dependent microautophagy pathway that 34 preferentially sorts aberrant proteasomes into the vacuole, thereby biasing accumulation of 35 functional proteasomes in PSGs. The proteasome core particle (CP) and regulatory particle (RP) 36 are regulated differently. Without AMPK, the insoluble protein deposit (IPOD) serves as an 37 alternative site that specifically sequesters CP aggregates. Our findings reveal a novel AMPK- 38 controlled ESCRT-mediated microautophagy mechanism in the regulation of proteasome 39 trafficking and homeostasis under carbon starvation. 40 41 42 43 44 45 46 47 2 bioRxiv preprint doi: https://doi.org/10.1101/751420; this version posted August 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 48 Introduction 49 The ubiquitin-proteasome system (UPS) is a conserved proteolytic system responsible for 50 the highly selective degradation of cellular proteins. Conjugation of ubiquitin to substrates targets 51 them to the proteasome for degradation [1, 2]. The 26S proteasome comprises a 20S core particle 52 (CP) with a 19S regulatory particle (RP) on one or both ends of the CP [1]. In the CP, four stacked 53 rings are assembled from different β-subunits (β1-β7) and α-subunits (α1-α7). The RP is 54 assembled from two multisubunit subcomplexes termed the base and lid [1]. The RP is responsible 55 for substrate binding, deubiquitylation, unfolding, and translocation [1, 3-5]. 56 The UPS is responsible for about 80-90% of cellular proteolysis under normal growth 57 conditions, and therefore, alterations in proteasome activity have a major impact on protein 58 homeostasis (“proteostasis”) [6, 7]. For instance, the age-related decline of proteostasis can be 59 compensated by increasing proteasome activity, while cancer cell growth can be limited by 60 inhibiting proteasome activity [8-10]. One way to regulate the availability of proteasomes is by 61 formation of Proteasome Storage Granules (PSGs), which are phase-separated cytoplasmic 62 structures that collect proteasomes during specific stresses. PSGs most likely serve as an adaptive 63 mechanism to regulate proteasome activity [11, 12]. Multiple factors have been reported to be 64 relevant to the assembly and disassembly of PSGs, including carbon starvation [13], intracellular 65 pH [14], protein N-α-acetylation [11], the activity of Blm10 [15], and the integrity of certain 66 proteasome lid subunits [12, 16, 17]. Nevertheless, relatively little is known about how proteasome 67 nucleocytoplasmic trafficking and homeostasis is regulated, particularly under carbon starvation 68 conditions. 69 Autophagy is another major means of degrading cellular components, which protects cells 70 from damaged proteins and organelles and promotes cell survival under various stresses, such as 3 bioRxiv preprint doi: https://doi.org/10.1101/751420; this version posted August 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 71 starvation, oxidative stress, protein aggregation, and ER stress. The cellular components of 72 macroautophagy have been extensively studied in yeast and assigned roles in selective or 73 nonselective macroautophagy or both [18]. All forms of macroautophagy share a common set of 74 core autophagy (Atg) proteins that are involved in autophagosome initiation and formation [19]. 75 Other Atg factors are required for specific types of selective macroautophagy [18]. Atg17 is 76 required only for nonselective macroautophagy [20]. 77 In comparison to macroautophagy, microautophagy is poorly understood [21]. Recently, 78 endosomal sorting complexes required for transport (ESCRT)- and clathrin-dependent 79 microautophagy has been described in yeast undergoing diauxic shift; the substrate protein 80 followed in this study was the transmembrane vacuolar protein Vph1 [22]. The ESCRT machinery 81 is an ancient system for membrane remodeling and scission; in eukaryotes it includes five distinct 82 subcomplexes ESCRT-0, I, II, III, and the AAA ATPase Vps4 [23]. The ESCRT pathway drives 83 diverse cellular processes, such as multivesicular body (MVB) formation, nuclear pore complex 84 (NPC) quality control, virus budding, viral replication-complex assembly, macroautophagy, and 85 microautophagy [23, 24]. How the ESCRT machinery is activated and regulated in 86 microautophagy, however, is unclear. 87 Proteasome homeostasis involves a balance between proteasome assembly and degradation. 88 Normally, proteasomes are very stable entities within the cell [25], but their degradation by 89 autophagy is induced under certain conditions. Proteasome degradation by macroautophagy 90 (“proteaphagy”) occurs in response to nitrogen starvation and has been described in yeast, plants, 91 and mammals [26-30]. A recent study reported that PSGs protect proteasomes from autophagic 92 degradation during carbon starvation [31], suggesting an physiological connection between PSGs 4 bioRxiv preprint doi: https://doi.org/10.1101/751420; this version posted August 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 93 and autophagy. It is unknown how the partitioning of proteasomes between assembling PSGs in 94 the cytoplasm and proteolysis in the vacuole is regulated. 95 AMP-activated protein kinase (AMPK) is a highly conserved regulator of energy 96 homeostasis in eukaryotes. The AMPK heterotrimeric complex is composed of a catalytic α 97 subunit (called Snf1 in S. cerevisiae) and two regulatory subunits: a β subunit (Sip1, Sip2, or Gal83) 98 and a γ subunit (Snf4) [32]. The AMPK pathway is activated when energy stores are depleted, 99 which modulates the switch from fermentation to respiration in yeast [32, 33]. Moreover, AMPK 100 coordinates a wide range of cellular processes, such as cell growth, autophagy, metabolism, cell 101 polarity, and cytoskeletal dynamics [34]. 102 In this study, we demonstrate that AMPK, the ESCRT machinery, and the insoluble protein 103 deposit (IPOD) function together in the regulation of proteasome trafficking and degradation under 104 glucose starvation. We show that cells utilize AMPK- and ESCRT-dependent microautophagy to 105 clear aberrant proteasomes through vacuolar proteolysis under these conditions and thus safeguard 106 reversible PSG assembly and the maintenance of functional proteasomes during glucose starvation. 107 Our cytological data suggest very high levels of microautophagy can occur under these conditions. 108 We find that proteasomes dissociate into CP and RP complexes that are regulated through distinct 109 trafficking mechanisms during glucose starvation. Furthermore, irreversible CP aggregates 110 accumulate if AMPK is inactivated and cells are carbon starved. In such mutants, irreversible CP 111 aggregates concentrate in the IPOD compartment. These findings
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