A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae

Acetic acid is inhibitory to the growth of the yeast Saccharomyces cerevisiae , causing ATP starvation and oxidative stress, which leads to the suboptimal production of fuels and chemicals from lignocellulosic biomass. In this study, where each strain of a CRISPRi library was characterized individually, many essential and respiratory growth-essential genes that regulate tolerance to acetic acid were identified, providing a new understanding of the stress response of yeast and new targets for the bioengineering of industrial yeast.

Proteasome Regulation by Reversible Tyrosine Phosphorylation at the Membrane

Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine (pTyr) sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation. Multiple receptor tyrosine kinases (RTKs) can trigger Rpt2-Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates 26S proteasome integrity and activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase non-receptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anti-cancer therapies.

Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function

The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.

Phosphatase UBLCP1 controls proteasome assembly

Ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1), an FCP/SCP phosphatase family member, was identified as the first proteasome phosphatase. UBLCP1 binds to proteasome subunit Rpn1 and dephosphorylates the proteasome in vitro . However, it is still unclear which proteasome subunit(s) are the bona fide substrate(s) of UBLCP1 and the precise mechanism for proteasome regulation remains elusive. Here, we show that UBLCP1 selectively binds to the 19S regulatory particle (RP) through its interaction with Rpn1, but not the 20S core particle (CP) or the 26S proteasome holoenzyme. In the RP, UBLCP1 dephosphorylates the subunit Rpt1, impairs its ATPase activity, and consequently disrupts the 26S proteasome assembly, yet it has no effects on the RP assembly from precursor complexes. The Rpn1-binding and phosphatase activities of UBLCP1 are essential for its function on Rpt1 dephosphorylation and proteasome activity both in vivo and in vitro . Our study establishes the essential role of the UBLCP1/Rpn1/Rpt1 complex in regulating proteasome assembly.

Unveiling Protein Kinase A Targets in Cryptococcus neoformans Capsule Formation

ABSTRACT The protein kinase A (PKA) signal transduction pathway has been associated with pathogenesis in many fungal species. Geddes and colleagues [mBio 7(1):e01862-15, 2016, doi:10.1128/mBio.01862-15] used quantitative proteomics approaches to define proteins with altered abundance during protein kinase A (PKA) activation and repression in the opportunistic human fungal pathogen Cryptococcus neoformans . They observed an association between microbial PKA signaling and ubiquitin-proteasome regulation of protein homeostasis. Additionally, they correlated these processes with expression of polysaccharide capsule on the fungal cell surface, the main virulence-associated phenotype in this organism. Not only are their findings important for microbial pathogenesis, but they also support similar associations between human PKA signaling and ubiquitinated protein accumulation in neurodegenerative diseases.