Title

Insights into the Regulation and Mechanism of 26S Proteasome Base Assembly in Saccharomyces cerevisiae

Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Hochstrasser, Mark

Abstract

In eukaryotes, the 19S proteasome base contains a heterohexameric ring that is made up of six distinct AAA+ ATPase proteins called Rpt1-Rpt6. In contrast, the archaeal proteasome base is composed of only a single type AAA+ ATPase protein called the proteasome-activating nucleotidase (PAN) that forms a homohexameric ring. A crystal structure of the N-terminal part of PAN in Methanocaldococcus jannaschii a trimer-of-dimers arrangement that is dependent on the alternating cis-trans configuration of the peptide bond preceding a conserved proline residue (P91) located between the coiled coil (CC) and oligonucleotide/oligosaccharide-binding (OB) domains. This proline is also found in Rpt1(P96), Rpt2(P103), Rpt3(P93), and Rpt5(P76) in Saccharomyces cerevisiae. The importance of these Rpt prolines in eukaryotic proteasome assembly was unknown.I found that this 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. Because Rpt2, Rpt3 and Rpt5 belong to distinct dimer pairs (Rpt1-Rpt2, Rpt3-Rpt6 and Rpt4-Rpt5) that coalesce to form the heterohexameric ring, this conservation pattern is consistent with the hypothesis that these are the “cis” subunits of the ring. However, I found that none of the single proline mutations (to alanine) was obviously deleterious to yeast growth. On the other hand, rpt2-P103A, rpt3-P96A, and rpt5-P76A all exhibited synthetic growth defects when combined with deletions of specific base assembly chaperones, but rpt1-P96A did not. The Rpt5-P76A mutation decreases the levels of the subunit and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant, uniquely among the double mutants, 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 chaperone-mediated protein quality control (PQC) machinery. In addition, I found that Not4 E3 ligase ubiquitinates Rpt5 subunit in this mutant and this ubiquitination event in turn inhibits proteasome assembly. I propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization. To discover novel regulators of proteasome assembly, I utilized a high-copy tiled yeast genomic library to identify dosage suppressors of the temperature sensitivity of an rpt2,5PA mutant strain. From my screen, I identified Nst1, a 142-kDa protein that when overexpressed, suppressed the temperature sensitivity and proteasome assembly defects of several base mutants. I observed a significant decrease in cytosolic aggregates of GFP-Rpt1 and GFP-Rpt6 in nas6∆ rpn14∆ cells, which lack two Rpt assembly chaperones, when Nst1 was overexpressed. Nst1 is a highly charged and polar protein predicted to have numerous disordered regions, characteristics commonly found in proteins that are able to segregate into subcellular condensates. In agreement with this, I found that both endogenous and overexpressed Nst1 can form cytosolic puncta that co-localize with processing body (P-body) components. Based on these findings, I hypothesized that Nst1 might be involved in sequestering mRNAs in P-bodies and, thereby, promoting their translational repression. Translational inhibition was previously reported to suppress aggregation and proteasome assembly defects in base mutants under heat stress. Indeed, I found that Nst1 overexpression inhibited global protein translation in the nas6∆ rpn14∆ proteasome assembly mutant strain. Additionally, I discovered that overexpression of several known P-body components was also able to suppress the aforementioned defects in nas6∆ rpn14∆. Taken together, my data indicate that Nst1 is a previously overlooked P-body component that plays a role in translational repression and when overexpressed, prevents sequestration of Rpt subunits into aggregates and rescues proteasome assembly under heat stress.

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