Summary: Levels of the ribosome, the conserved molecular machine that mediates translation, are tightly linked to cellular growth rate. In humans, ribosomopathies are diseases associated with cell-type-specific pathologies and reduced ribosomal protein (RP) levels. Because gene expression defects resulting from ribosome deficiency have not yet been experimentally defined, we systematically probed mRNA, translation, and protein signatures that were either unlinked or linked to cellular growth rate in RP-deficient yeast cells. Ribosome deficiency was associated with altered translation of gene sub-classes, and profound general secondary effects of RP loss on the spectrum of cellular mRNAs were seen. Among these effects, growth-defective 60S mutants increased synthesis of proteins involved in proteasome-mediated degradation, whereas 40S mutants accumulated mature 60S subunits and increased translation of ribosome biogenesis genes. These distinct signatures of protein synthesis suggest intriguing and currently mysterious differences in the cellular consequences of deficiency for small and large ribosomal subunits.

Cellular stress responses often require transcription-based activation of gene expression to promote cellular adaptation. Whether general mechanisms exist for stress-responsive gene downregulation is less clear. A recently defined mechanism enables both up- and downregulation of protein levels for distinct gene sets by the same transcription factor via coordinated induction of canonical mRNAs and long undecoded transcript isoforms (LUTIs). We analyzed parallel gene expression datasets to determine whether this mechanism contributes to the conserved Hac1-driven branch of the unfolded protein response (UPR), indeed observing Hac1-dependent protein downregulation accompanying the upregulation of ER-related proteins that typifies UPR activation. Proteins downregulated by Hac1-driven LUTIs include those with electron transport chain (ETC) function. Abrogated ETC function improves the fitness of UPR-activated cells, suggesting functional importance to this regulation. We conclude that the UPR drives large-scale proteome remodeling, including coordinated up- and downregulation of distinct protein classes, which is partly mediated by Hac1-induced LUTIs.

SUMMARY Protein degradation is known to be a key component of expression regulation for individual genes, but its global impact on gene expression has been difficult to determine. We analyzed a parallel gene expression dataset of yeast meiotic differentiation, identifying instances of coordinated protein-level decreases to identify new cases of regulated meiotic protein degradation, including of ribosomes and targets of the meiosis-specific anaphase-promoting complex adaptor Ama1. Comparison of protein and translation measurements over time also revealed that, although meiotic cells are capable of synthesizing protein complex members at precisely matched levels, they typically do not. Instead, the members of most protein complexes are synthesized imprecisely, but their protein levels are matched, indicating that wild-type eukaryotic cells routinely use post-translational adjustment of protein complex partner levels to achieve proper stoichiometry. Outlier cases, in which specific complex components show divergent protein-level trends, suggest timed regulation of these complexes.

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The diacylglycerol‐regulated novel protein kinase C theta is highly druggable, but how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Cancer‐associated mutations in Ca2+‐regulated (conventional) PKC isozymes are generally loss‐of‐function, reframing PKC as having a tumor suppressive property, but whether this is the case for PKC Theta (θ) has not been addressed. Here we examine several cancer‐associated mutations in PKCθ, focusing on those at domain interfaces that are likely to impair autoinhibition, to begin to explore whether this isozyme of the PKC family functions to promote or suppress oncogenic signaling. PKCθ is selectively expressed in hematopoietic cells, namely platelets and T lymphocytes, where it regulates inflammatory signaling. Unsurprisingly, deregulated PKCθ has been implicated in many diseases including cancer where dozens of mutations have already been identified. One such residue was R145 in the autoinhibitory pseudosubstrate segment of PKCθ, whose mutation to His or Cys has been reported for two different cancers. This Arg interacts with Asp465 and Asp508 in the substrate binding cavity to maintain effective autoinhibition in the absence of the allosteric activator, diacylglycerol. We determined the effect of R145H and R145C mutation on the cellular activity and stability of PKCθ. Specifically, we used a genetically‐encoded FRET‐based reporter, C kinase activity reporter 2 (CKAR2) to measure the basal and agonist‐evoked activity of PKCθ. Whereas wild‐type PKCθ was effectively autoinhibited and required addition of agonist (here the phorbol ester, PDBu) for activation, both the R145H and R145C mutants were almost maximally activated under basal conditions. However, addition of cycloheximide to inhibit protein synthesis revealed that the half‐time of turn‐over of the mutants was 3‐ and 8‐fold faster than that of wild‐type enzyme, respectively. Taken together, these data suggest that the R145H and R145C mutants fail to autoinhibit resulting in a PKC with constitutive activity that is unstable and subject to the quality‐control degradation we have previously reported for Ca2+‐regulated PKC isozymes. Thus, these pseudosubstrate mutations in PKCθ are paradoxically loss‐of‐function because the constitutively active kinases are sensitive to degradation.