The heat shock protein LarA activates the Lon protease in response to proteotoxic stress.

The Lon protease is a highly conserved protein degradation machine that has critical regulatory and protein quality control functions in cells from the three domains of life. Here, we report the discovery of a α-proteobacterial heat shock protein, LarA, that functions as a dedicated Lon regulator. We show that LarA accumulates at the onset of proteotoxic stress and allosterically activates Lon-catalysed degradation of a large group of substrates through a five amino acid sequence at its C-terminus. Further, we find that high levels of LarA cause growth inhibition in a Lon-dependent manner and that Lon-mediated degradation of LarA itself ensures low LarA levels in the absence of stress. We suggest that the temporal LarA-dependent activation of Lon helps to meet an increased proteolysis demand in response to protein unfolding stress. Our study defines a regulatory interaction of a conserved protease with a heat shock protein, serving as a paradigm of how protease activity can be tuned under changing environmental conditions.

Regulation of the general stress response sigma factor σT by Lon-mediated proteolysis.

IMPORTANCE: Regulated protein degradation is a critical process in all cell types, which contributes to the precise regulation of protein amounts in response to internal and external cues. In bacteria, protein degradation is carried out by ATP-dependent proteases. Although past work revealed detailed insights into the operation principles of these proteases, there is limited knowledge about the substrate proteins that are degraded by distinct proteases and the regulatory role of proteolysis in cellular processes. This study reveals a direct role of the conserved protease Lon in regulating σT, a transcriptional regulator of the general stress response in α-proteobacteria. Our work is significant as it underscores the importance of regulated proteolysis in modulating the levels of key regulatory proteins under changing conditions.

Tricalbin proteins regulate plasma membrane phospholipid homeostasis.

The evolutionarily conserved extended synaptotagmin (E-Syt) proteins are calcium-activated lipid transfer proteins that function at contacts between the ER and plasma membrane (ER-PM contacts). However, roles of the E-Syt family members in PM lipid organisation remain incomplete. Among the E-Syt family, the yeast tricalbin (Tcb) proteins are essential for PM integrity upon heat stress, but it is not known how they contribute to PM maintenance. Using quantitative lipidomics and microscopy, we find that the Tcb proteins regulate phosphatidylserine homeostasis at the PM. Moreover, upon heat-induced membrane stress, Tcb3 co-localises with the PM protein Sfk1 that is implicated in PM phospholipid asymmetry and integrity. The Tcb proteins also control the PM targeting of the known phosphatidylserine effector Pkc1 upon heat-induced stress. Phosphatidylserine has evolutionarily conserved roles in PM organisation, integrity, and repair. We propose that phospholipid regulation is an ancient essential function of E-Syt family members required for PM integrity.

The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle.

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Δlon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

A heat-sensitive Osh protein controls PI4P polarity.

BACKGROUND: Phosphoinositide lipids provide spatial landmarks during polarized cell growth and migration. Yet how phosphoinositide gradients are oriented in response to extracellular cues and environmental conditions is not well understood. Here, we elucidate an unexpected mode of phosphatidylinositol 4-phosphate (PI4P) regulation in the control of polarized secretion. RESULTS: We show that PI4P is highly enriched at the plasma membrane of growing daughter cells in budding yeast where polarized secretion occurs. However, upon heat stress conditions that redirect secretory traffic, PI4P rapidly increases at the plasma membrane in mother cells resulting in a more uniform PI4P distribution. Precise control of PI4P distribution is mediated through the Osh (oxysterol-binding protein homology) proteins that bind and present PI4P to a phosphoinositide phosphatase. Interestingly, Osh3 undergoes a phase transition upon heat stress conditions, resulting in intracellular aggregates and reduced cortical localization. Both the Osh3 GOLD and ORD domains are sufficient to form heat stress-induced aggregates, indicating that Osh3 is highly tuned to heat stress conditions. Upon loss of Osh3 function, the polarized distribution of both PI4P and the exocyst component Exo70 are impaired. Thus, an intrinsically heat stress-sensitive PI4P regulatory protein controls the spatial distribution of phosphoinositide lipid metabolism to direct secretory trafficking as needed. CONCLUSIONS: Our results suggest that control of PI4P metabolism by Osh proteins is a key determinant in the control of polarized growth and secretion.

Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor-controlled Ssy5 signaling protease.

The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5) sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy terminus of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Δtether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution, and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non- and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.

Regulation of the replication initiator DnaA in Caulobacter crescentus.

The decision to initiate DNA replication is a critical step in the cell cycle of all organisms. In nearly all bacteria, replication initiation requires the activity of the conserved replication initiation protein DnaA. Due to its central role in cell cycle progression, DnaA activity must be precisely regulated. This review summarizes the current state of DnaA regulation in the asymmetrically dividing α-proteobacterium Caulobacter crescentus, an important model for bacterial cell cycle studies. Mechanisms will be discussed that regulate DnaA activity and abundance under optimal conditions and in coordination with the asymmetric Caulobacter cell cycle. Furthermore, we highlight recent findings of how regulated DnaA synthesis and degradation collaborate to adjust DnaA abundance under stress conditions. The mechanisms described provide important examples of how DNA replication is regulated in an α-proteobacterium and thus represent an important starting point for the study of DNA replication in many other bacteria. This article is part of a Special Issue entitled: Dynamic gene expression, edited by Prof. Patrick Viollier.

Phosphoinositide kinase signaling controls ER-PM cross-talk.

Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca(2+)-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase-mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions.

Protein quality control at the inner nuclear membrane.

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron.

The Ssy1-Ptr3-Ssy5 (SPS)-sensing pathway enables yeast to respond to extracellular amino acids. Stp1, the effector transcription factor, is synthesized as a latent cytoplasmic precursor with an N-terminal regulatory domain that restricts its nuclear accumulation. The negative regulatory mechanisms impinging on the N-terminal domain are poorly understood. However, Stp1 latency depends on three inner nuclear membrane proteins, Asi1, Asi2, and Asi3. We report that the N-terminal domain of Stp1 contains a small motif, designated RI, that fully accounts for latency. RI is modular, mediates interactions with the plasma membrane, and can retain histone Htb2 in the cytoplasm. A novel class of STP1 mutations affecting RI were isolated that are less efficiently retained in the cytoplasm but remain under tight negative control by the Asi proteins. Intriguingly, these mutant proteins exhibit enhanced stability in strains lacking ASI1. Our results indicate that RI mediates latency by two distinct activities: it functions as a cytoplasmic retention determinant and an Asi-dependent degron. These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the inner nuclear membrane Asi proteins function in a degradation pathway in the nucleus.

Rts1-protein phosphatase 2A antagonizes Ptr3-mediated activation of the signaling protease Ssy5 by casein kinase I.

Ligand-induced conformational changes of plasma membrane receptors initiate signals that enable cells to respond to discrete extracellular cues. In response to extracellular amino acids, the yeast Ssy1-Ptr3-Ssy5 sensor triggers the endoproteolytic processing of transcription factors Stp1 and Stp2 to induce amino acid uptake. Activation of the processing protease Ssy5 depends on the signal-induced phosphorylation of its prodomain by casein kinase I (Yck1/2). Phosphorylation is required for subsequent Skp1/Cullin/Grr1 E3 ubiquitin ligase-dependent polyubiquitylation and proteasomal degradation of the inhibitory prodomain. Here we show that Rts1, a regulatory subunit of the general protein phosphatase 2A, and Ptr3 have opposing roles in controlling Ssy5 prodomain phosphorylation. Rts1 constitutively directs protein phosphatase 2A activity toward the prodomain, effectively setting a signaling threshold required to mute Ssy5 activation in the absence of amino acid induction. Ptr3 functions as an adaptor that transduces conformational signals initiated by the Ssy1 receptor to dynamically induce prodomain phosphorylation by mediating the proximity of the Ssy5 prodomain and Yck1/2. Our results demonstrate how pathway-specific and general signaling components function synergistically to convert an extracellular stimulus into a highly specific, tuned, and switch-like transcriptional response that is critical for cells to adapt to changes in nutrient availability.

A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy5.

Regulated proteolysis serves as a mechanism to control cellular processes. The SPS (Ssy1-Ptr3-Ssy5) sensor in yeast responds to extracellular amino acids by endoproteolytically activating transcription factors Stp1 and Stp2 (Stp1/2). The processing endoprotease Ssy5 is regulated via proteasomal degradation of its noncovalently associated N-terminal prodomain. We find that degradation of the prodomain requires a conserved phosphodegron comprising phosphoacceptor sites and ubiquitin-accepting lysine residues. Upon amino acid induction, the phosphodegron is modified in a series of linked events by a set of general regulatory factors involved in diverse signaling pathways. First, an amino acid-induced conformational change triggers phosphodegron phosphorylation by the constitutively active plasma membrane-localized casein kinase I (Yck1/2). Next the prodomain becomes a substrate for polyubiquitylation by the Skp1/Cullin/Grr1 E3 ubiquitin ligase complex (SCF(Grr1)). Finally, the modified prodomain is concomitantly degraded by the 26S proteasome. These integrated events are requisite for unfettering the Ssy5 endoprotease, and thus Stp1/2 processing. The Ssy5 phosphoacceptor motif resembles the Yck1/2- and Grr1-dependent degrons of regulators in the Snf3/Rgt2 glucose-sensing pathway. Our work defines a novel proteolytic activation cascade that regulates an intracellular signaling protease and illustrates how general signaling components are recruited to distinct pathways that achieve conditional and specific signaling outputs.

The prodomain of Ssy5 protease controls receptor-activated proteolysis of transcription factor Stp1.

Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells.