Diverse Sphingolipid Species Harbor Different Effects on Ire1 Clustering.

Endoplasmic reticulum (ER) function is dedicated to multiple essential processes in eukaryotes, including the processing of secretory proteins and the biogenesis of most membrane lipids. These roles implicate a heavy burden to the organelle, and it is thus prone to fluctuations in the homeostasis of molecules which govern these processes. The unfolded protein response (UPR) is a general ER stress response tasked with maintaining the ER for optimal function, mediated by the master activator Ire1. Ire1 is an ER transmembrane protein that initiates the UPR, forming characteristic oligomers in response to irregularities in luminal protein folding and in the membrane lipid environment. The role of lipids in regulating the UPR remains relatively obscure; however, recent research has revealed a potent role for sphingolipids in its activity. Here, we identify a major role for the oxysterol-binding protein Kes1, whose activity is of consequence to the sphingolipid profile in cells resulting in an inhibition of UPR activity. Using an mCherry-tagged derivative of Ire1, we observe that this occurs due to inhibition of Ire1 to form oligomers. Furthermore, we identify that a sphingolipid presence is required for Ire1 activity, and that specific sphingolipid profiles are of major consequence to Ire1 function. In addition, we highlight cases where Ire1 oligomerization is absent despite an active UPR, revealing a potential mechanism for UPR induction where Ire1 oligomerization is not necessary. This work provides a basis for the role of sphingolipids in controlling the UPR, where their metabolism harbors a crucial role in regulating its onset.

Cancer associated mutations in Sec61γ alter the permeability of the ER translocase.

Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the Sec61 complex, a heterotrimeric protein complex possessing two essential sub-units, Sec61p/Sec61α and Sss1p/Sec61γ and the non-essential Sbh1p/Sec61ß subunit. In addition to forming a protein conducting channel, the Sec61 complex maintains the ER permeability barrier, preventing flow of molecules and ions. Loss of Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C-terminus is juxtaposed to the key gating module of Sec61p/Sec61α and is important for gating the translocon. Inspection of the cancer genome database identifies six mutations in highly conserved amino acids of Sec61γ/Sss1p. We identify that five out of the six mutations identified affect gating of the ER translocon, albeit with varying strength. Together, we find that mutations in Sec61γ that arise in malignant cells result in altered translocon gating dynamics, this offers the potential for the translocon to represent a target in co-therapy for cancer treatment.

The conserved C-terminus of Sss1p is required to maintain the endoplasmic reticulum permeability barrier.

The endoplasmic reticulum (ER) is the entry point to the secretory pathway and major site of protein biogenesis. Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the evolutionarily conserved Sec61 complex, a heterotrimeric channel that comprises the Sec61p/Sec61α, Sss1p/Sec61γ, and Sbh1p/Sec61ß subunits. In addition to forming a protein-conducting channel, the Sec61 complex also functions to maintain the ER permeability barrier, preventing the mass free flow of essential ER-enriched molecules and ions. Loss in Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C terminus is juxtaposed to the key gating module of Sec61p/Sec61α, and we hypothesize it is important for gating the ER translocon. The ER stress response was found to be constitutively induced in two temperature-sensitive sss1 mutants (sss1ts ) that are still proficient to conduct ER translocation. A screen to identify intergenic mutations that allow for sss1ts cells to grow at 37 °C suggests the ER permeability barrier to be compromised in these mutants. We propose the extreme C terminus of Sss1p/Sec61γ is an essential component of the gating module of the ER translocase and is required to maintain the ER permeability barrier.

Functional diversification of the chemical landscapes of yeast Sec14-like phosphatidylinositol transfer protein lipid-binding cavities.

Phosphatidylinositol-transfer proteins (PITPs) are key regulators of lipid signaling in eukaryotic cells. These proteins both potentiate the activities of phosphatidylinositol (PtdIns) 4-OH kinases and help channel production of specific pools of phosphatidylinositol 4-phosphate (PtdIns(4)P) dedicated to specific biological outcomes. In this manner, PITPs represent a major contributor to the mechanisms by which the biological outcomes of phosphoinositide are diversified. The two-ligand priming model proposes that the engine by which Sec14-like PITPs potentiate PtdIns kinase activities is a heterotypic lipid-exchange cycle where PtdIns is a common exchange substrate among the Sec14-like PITP family, but the second exchange ligand varies with the PITP. A major prediction of this model is that second-exchangeable ligand identity will vary from PITP to PITP. To address the heterogeneity in the second exchange ligand for Sec14-like PITPs, we used structural, computational, and biochemical approaches to probe the diversities of the lipid-binding cavity microenvironments of the yeast Sec14-like PITPs. The collective data report that yeast Sec14-like PITP lipid-binding pockets indeed define diverse chemical microenvironments that translate into differential ligand-binding specificities across this protein family.

An equal opportunity collaboration between lipid metabolism and proteins in the control of membrane trafficking in the trans-Golgi and endosomal systems.

Recent years have witnessed the evolution of the cell biology of lipids into an extremely active area of investigation. Deciphering the involvement of lipid metabolism and lipid signaling in membrane trafficking pathways defines a major nexus of contemporary experimental activity on this front. Significant effort in that direction is invested in understanding the trans-Golgi network/endosomal system where unambiguous connections between membrane trafficking and inositol lipid and phosphatidylcholine metabolism were first discovered. However, powered by new advances in contemporary cell biology, the march of science is rapidly expanding that window of inquiry to include ever more diverse arms of the lipid metabolome, and to include other compartments of the secretory pathway as well.

WITHDRAWN: The Fundamental And Pathological Importance Of Oxysterol Binding Protein And Its Related Proteins.

This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98.

A Lipid Transfer Protein Signaling Axis Exerts Dual Control of Cell-Cycle and Membrane Trafficking Systems.

Kes1/Osh4 is a member of the conserved, but functionally enigmatic, oxysterol binding protein-related protein (ORP) superfamily that inhibits phosphatidylinositol transfer protein (Sec14)-dependent membrane trafficking through the trans-Golgi (TGN)/endosomal network. We now report that Kes1, and select other ORPs, execute cell-cycle control activities as functionally non-redundant inhibitors of the G1/S transition when cells confront nutrient-poor environments and promote replicative aging. Kes1-dependent cell-cycle regulation requires the Greatwall/MASTL kinase ortholog Rim15, and is opposed by Sec14 activity in a mechanism independent of Kes1/Sec14 bulk membrane-trafficking functions. Moreover, the data identify Kes1 as a non-histone target for NuA4 through which this lysine acetyltransferase co-modulates membrane-trafficking and cell-cycle activities. We propose the Sec14/Kes1 lipid-exchange protein pair constitutes part of the mechanism for integrating TGN/endosomal lipid signaling with cell-cycle progression and hypothesize that ORPs define a family of stage-specific cell-cycle control factors that execute tumor-suppressor-like functions.

The Impact of Cooling Rate on the Safety of Food Products as Affected by Food Containers.

In recent decades, the demand for ready-to-eat (RTE) food items prepared by the food catering sector has increased together with the value of cook-serve, cook-chill, and cook-freeze food products. The technologies by which foods are cooked, chilled, refrigerated for storage, and reheated before serving are of prime importance to maintain safety. Packaging materials and food containers play an important role in influencing the cooling rate of RTE foods. Food items that are prepared using improper technologies and inappropriate packaging materials may be contaminated with foodborne pathogens. Numerous research studies have shown the impact of deficient cooling technologies on the survival and growth of foodborne pathogens, which may subsequently pose a threat to public health. The operating temperatures and cooling rates of the cooling techniques applied must be appropriate to inhibit the growth of pathogens. Food items must be stored outside the temperature danger zone, which is between 5 and 60 °C, in order to inhibit the growth of these pathogens. The cooling techniques used to prepare potentially hazardous foods, such as cooked meat, rice, and pasta, must be properly applied and controlled to ensure food safety. This paper critically reviews the effects of cooling and its relationship to food containers on the safety of RTE foods produced and sold through the food service industry.

Diminished Ost3-dependent N-glycosylation of the BiP nucleotide exchange factor Sil1 is an adaptive response to reductive ER stress.

BiP (Kar2 in yeast) is an essential Hsp70 chaperone and master regulator of endoplasmic reticulum (ER) function. BiP's activity is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, Sil1 and Lhs1. Both Sil1 and Lhs1 are glycoproteins, but how N-glycosylation regulates their function is not known. Here, we show that N-glycosylation of Sil1, but not of Lhs1, is diminished upon reductive stress. N-glycosylation of Sil1 is predominantly Ost3-dependent and requires a functional Ost3 CxxC thioredoxin motif. N-glycosylation of Lhs1 is largely Ost3-independent and independent of the CxxC motif. Unglycosylated Sil1 is not only functional but is more effective at rescuing loss of Lhs1 activity than N-glycosylated Sil1. Furthermore, substitution of the redox active cysteine pair C52 and C57 in the N terminus of Sil1 results in the Doa10-dependent ERAD of this mutant protein. We propose that reductive stress in the ER inhibits the Ost3-dependent N-glycosylation of Sil1, which regulates specific BiP functions appropriate to the needs of the ER under reductive stress.

Lipid transfer proteins and the tuning of compartmental identity in the Golgi apparatus.

The Golgi complex constitutes a central way station of the eukaryotic endomembrane system, an intricate network of organelles engaged in control of membrane trafficking and the processing of various cellular components. Previous ideas of compartmental stability within this network are gradually being reshaped by concepts describing a biochemical continuum of hybrid organelles whose constitution is regulated by compartmental maturation. Membrane lipid composition and lipid signaling processes make fundamental contributions to compartmentalization strategies that are themselves critical for organizing cellular architecture and biochemical activities. Phosphatidylinositol transfer proteins (PITPs) are increasingly recognized as key regulators of membrane trafficking through the secretory pathway. They do so by coordinating lipid metabolism with lipid signaling, translating this information to core protein components of the membrane trafficking machinery. In this capacity, PITPs can be viewed as regulators of an essential lipid-protein interface of cisternal maturation. It is also now becoming appreciated, for the first time, that such an interface plays important roles in larger systems processes that link secretory pathway function with cell proliferation.

Dissecting the role of histidine kinase and HOG1 mitogen-activated protein kinase signalling in stress tolerance and pathogenicity of Parastagonospora nodorum on wheat.

The HOG1 mitogen-activated protein kinase (MAPK) pathway is activated through two-component histidine kinase (HK) signalling. This pathway was first characterized in the budding yeast Saccharomyces cerevisiae as a regulator of osmotolerance. The fungus Parastagonospora nodorum is the causal agent of septoria nodorum blotch of wheat. This pathogen uses host-specific effectors in tandem with general pathogenicity mechanisms to carry out its infection process. Genes showing strong sequence homology to S. cerevisiae HOG1 signalling pathway genes have been identified in the genome of P. nodorum. In this study, we examined the role of the pathway in the virulence of P. nodorum on wheat by disrupting putative pathway component genes: HOG1 (SNOG_13296) MAPK and NIK1 (SNOG_11631) hybrid HK. Mutants deleted in NIK1 and HOG1 were insensitive to dicarboximide and phenylpyrrole fungicides, but not a fungicide that targets ergosterol biosynthesis. Furthermore, both Δnik1 and Δhog1 mutants showed increased sensitivity to hyperosmotic stress. However, HOG1, but not NIK1, is required for tolerance to elevated temperatures. HOG1 deletion conferred increased tolerance to 6-methoxy-2-benzoxazolinone, a cereal phytoalexin. This suggests that the HOG1 signalling pathway is not exclusively associated with NIK1. Both Δnik1 and Δhog1 mutants retained the ability to infect and cause necrotic lesions on wheat. However, we observed that the Δhog1 mutation resulted in reduced production of pycnidia, asexual fruiting bodies that facilitate spore dispersal during late infection. Our study demonstrated the overlapping and distinct roles of a HOG1 MAPK and two-component HK signalling in P. nodorum growth and pathogenicity.

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis.

Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.

PITPs as targets for selectively interfering with phosphoinositide signaling in cells.

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs nor PITPs in general have been exploited as targets for chemical inhibition for such purposes. Herein, we validate what is to our knowledge the first small-molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs) and are effective inhibitors in vitro and in vivo. We further establish that Sec14 is the sole essential NPPM target in yeast and that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects and demonstrate that NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof of concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies.

Golgi membrane dynamics and lipid metabolism.

The striking morphology of the Golgi complex has fascinated cell biologists since its discovery over 100 years ago. Yet, despite intense efforts to understand how membrane flow relates to Golgi form and function, this organelle continues to baffle cell biologists and biochemists alike. Fundamental questions regarding Golgi function, while hotly debated, remain unresolved. Historically, Golgi function has been described from a protein-centric point of view, but we now appreciate that conceptual frameworks for how lipid metabolism is integrated with Golgi biogenesis and function are essential for a mechanistic understanding of this fascinating organelle. It is from a lipid-centric perspective that we discuss the larger question of Golgi dynamics and membrane trafficking. We review the growing body of evidence for how lipid metabolism is integrally written into the engineering of the Golgi system and highlight questions for future study.

Local control of phosphatidylinositol 4-phosphate signaling in the Golgi apparatus by Vps74 and Sac1 phosphoinositide phosphatase.

In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (K(D) = 3.8 µM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents.

Sec14 like PITPs couple lipid metabolism with phosphoinositide synthesis to regulate Golgi functionality.

An interface coordinating lipid metabolism with proteins that regulate membrane trafficking is necessary to regulate Golgi morphology and dynamics. Such an interface facilitates the membrane deformations required for vesicularization, forms platforms for protein recruitment and assembly on appropriate sites on a membrane surface and provides lipid co-factors for optimal protein activity in the proper spatio-temporally regulated manner. Importantly, Sec14 and Sec14-like proteins are a unique superfamily of proteins that sense specific aspects of lipid metabolism, employing this information to potentiate phosphoinositide production. Therefore, Sec14 and Sec14 like proteins form central conduits to integrate multiple aspects of lipid metabolism with productive phosphoinositide signaling.

The oxysterol-binding protein superfamily: new concepts and old proteins.

The Kes1 OSBP (oxysterol-binding protein) is a key regulator of membrane trafficking through the TGN (trans-Golgi network) and endosomal membranes. We demonstrated recently that Kes1 acts as a sterol-regulated rheostat for TGN/endosomal phosphatidylinositol 4-phosphate signalling. Kes1 utilizes its dual lipid-binding activities to integrate endosomal lipid metabolism with TORC1 (target of rapamycin complex 1)-dependent proliferative pathways and transcriptional control of nutrient signalling.

A sterol-binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing.

Kes1, and other oxysterol-binding protein superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids and dampens gene expression driven by Gcn4, the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic "large Mediator" complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing.

Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution.

Sec14-superfamily proteins integrate the lipid metabolome with phosphoinositide synthesis and signaling via primed presentation of phosphatidylinositol (PtdIns) to PtdIns kinases. Sec14 action as a PtdIns-presentation scaffold requires heterotypic exchange of phosphatidylcholine (PtdCho) for PtdIns, or vice versa, in a poorly understood progression of regulated conformational transitions. We identify mutations that confer Sec14-like activities to a functionally inert pseudo-Sec14 (Sfh1), which seemingly conserves all of the structural requirements for Sec14 function. Unexpectedly, the "activation" phenotype results from alteration of residues conserved between Sfh1 and Sec14. Using biochemical and biophysical, structural, and computational approaches, we find the activation mechanism reconfigures atomic interactions between amino acid side chains and internal water in an unusual hydrophilic microenvironment within the hydrophobic Sfh1 ligand-binding cavity. These altered dynamics reconstitute a functional "gating module" that propagates conformational energy from within the hydrophobic pocket to the helical unit that gates pocket access. The net effect is enhanced rates of phospholipid-cycling into and out of the Sfh1* hydrophobic pocket. Taken together, the directed evolution approach reveals an unexpectedly flexible functional engineering of a Sec14-like PtdIns transfer protein-an engineering invisible to standard bioinformatic, crystallographic, and rational mutagenesis approaches.