Biology subjects

Hochstrasser, M.

Publications and source records attributed to Hochstrasser, M..

4 recordsLinked to original sources

bioRxiv2021

Selective Microautophagy of Proteasomes is Initiated by ESCRT-0 and Is Promoted by Proteasome Ubiquitylation

The proteasome is central to proteolysis by the ubiquitin-proteasome system under normal growth conditions but is itself degraded through macroautophagy under nutrient stress. A recently described AMPK (AMP-activated protein kinase)-regulated ESCRT (endosomal sorting complex required for transport)-dependent microautophagy pathway also regulates proteasome trafficking and degradation in low glucose conditions in yeast. Aberrant proteasomes are more prone to microautophagy, suggesting the ESCRT system fine-tunes proteasome quality control under low glucose stress. Here we uncover additional features of the selective microautophagy of proteasomes. Genetic or pharmacological induction of aberrant proteasomes is associated with increased mono- or oligo-ubiquitylation of proteasome components, which appear to be recognized by ESCRT-0. AMPK controls this pathway in part by regulating the trafficking of ESCRT-0 to the vacuole surface, which also leads to degradation of the Vps27 subunit of ESCRT-0. The Rsp5 ubiquitin ligase contributes to proteasome subunit ubiquitylation, and multiple ubiquitin-binding elements in Vps27 are involved in their recognition. We propose that ESCRT-0 at the vacuole surface recognizes ubiquitylated proteasomes and initiates their microautophagic elimination during glucose depletion. Saummary statementESCRT-0 selectively targets aberrant proteasomes for microautophagy by recognition of proteasome ubiquitylation status to fine-tune proteasome quality control under low glucose conditions.

cell biology↗

bioRxiv2020

Conserved prolines in the coiled coil-OB domain linkers of proteasomal ATPases facilitate eukaryotic proteasome base assembly

The proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.

biochemistry↗

bioRxiv2020

Crystal structure of a guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi

Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho-GTPases Rac1 and Cdc42. We discovered a new domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEF alone (3.0 [A]) and complexed with Rac1 (1.7 [A]) reveal striking convergent evolution, with a distinct topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a novel mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEF alone alters cell morphology. Cumulatively, this work reveals a novel bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to evoke changes in cytoskeletal structure.

microbiology↗

bioRxiv2019

AMPK regulates ESCRT-dependent microautophagy of proteasomes concomitant with proteasome storage granule assembly during glucose starvation

The ubiquitin-proteasome system regulates numerous cellular processes and is central to protein homeostasis. In proliferating yeast and many mammalian cells, proteasomes are highly enriched in the nucleus. In carbon-starved yeast, proteasomes migrate to the cytoplasm and collect in phase-separated proteasome storage granules (PSGs). PSGs dissolve and proteasomes return to the nucleus within minutes of glucose refeeding. The mechanisms by which cells regulate proteasome homeostasis under these conditions remain largely unknown. Here we show that AMP-activated protein kinase (AMPK) together with endosomal sorting complexes required for transport (ESCRTs) drive a glucose starvation-dependent microautophagy pathway that preferentially sorts aberrant proteasomes into the vacuole, thereby biasing accumulation of functional proteasomes in PSGs. The proteasome core particle (CP) and regulatory particle (RP) are regulated differently. Without AMPK, the insoluble protein deposit (IPOD) serves as an alternative site that specifically sequesters CP aggregates. Our findings reveal a novel AMPK-controlled ESCRT-mediated microautophagy mechanism in the regulation of proteasome trafficking and homeostasis under carbon starvation.

cell biology↗