The mechanism of cyclic proteasome-mediated protein degradation in Sulfolobus acidocaldarius

Abstract

Sulfolobus acidocaldarius, a member of the TACK archaea superphylum, is one of the closest, experimentally tractable Prokaryotic relatives of Eukaryotes. Importantly, like Eukaryotes, Sulfolobus cells maintain an ordered cell cycle, despite lacking eukaryotic-like cell cycle regulators (homologues of cyclin and cyclin-dependent kinases). This raises the question of how Sulfolobus cells control cell cycle progression. While this is not yet known, previous work by members of the Baum lab showed that proteasome-mediated protein degradation is required for the onset of cell division and at division for the initiation of a new round of DNA replication in the following cell cycle. Division was also associated with the rapid proteasome-mediated degradation of a non-contractile homologue of ESCRT-III proteins, CdvB (Cell division protein B), while the ESCRT-III homologues CdvB1 and CdvB2 remained stable until G1, enabling them to drive cytokinesis and abscission to completion. Thus, tight control over the timing and specificity of proteasome-mediated degradation plays a critical role in the division process in Sulfolobus. Building on this work, the goal of my thesis is to explore the mechanism of the regulation of cyclic proteolysis in Sulfolobus by studying the role of I) the ESCRT-III C terminal region in determining the relative stability of CdvB, B1 and B2, II) the proteasome, III) the PAN AAA+ ATP-ase, IV) Ubiquitin-like proteins in this process. In doing so, I show that C terminal region of CdvB is responsible for CdvB’s susceptibility to degradation at division. Further, my data suggest that the signal triggering CdvB is complex and combines basal degradation across the cycle, together with CdvA-mediated protection from degradation, and instability at division that depends on more than one sequence in the CdvB tail. Intriguingly, the mechanism by which proteasome-mediated degradation of CdvB occurs appear to be independent of ubiquitin-like modifiers or posttranslational modifications in general. However, overexpression of hydrolysis defective PAN led to retention of CdvB in both pre-constricting and constricting cells rings due to partial inhibition of degradation. These data lead us to suggest a model in which CdvB’s degradation at division depends on its interaction with its partners – CdvA, CdvB1/B2 and the AAA+ ATPase Vps4. When CdvB protein first accumulates at D phase entry, the broken wH domain of CdvB binds to CdvA to form a stable division ring. Once CdvB1 and B2 have been 8 assembled into a co-polymer, the disassembly of the CdvA ring exposes the MIM2 interaction motif of CdvB to Vps4, which drives the disassembly of CdvB polymers. In the absence of a CdvA ring, the broken wH in monomeric CdvB functions as a signal to promote rapid protein proteolysis via the proteasome. To conclude, these findings offer a deeper understanding of cell cycle control in archaea and point to the importance of proteasomal degradation in mediating the transition of G2 to G1.