Biology subjects

Stefan, C. J.

Publications and source records attributed to Stefan, C. J..

6 recordsLinked to original sources

bioRxiv2026

Ire1 membrane stress responses support cell growth upon disruptions in inter-organelle contacts

Disruptions in inter-organelle contacts result in membrane lipid homeostasis defects that ultimately impair cellular function and viability. Yet, essential responses to membrane lipid imbalances remain poorly understood. In this study, we demonstrate that Ire1-dependent Membrane Stress Responses (MSR), distinct from the canonical Unfolded Protein Response (UPR), sustain the growth of yeast cells lacking inter-organelle contacts. Comprehensive lipidomics reveal that the Ire1-mediated MSR compensates for glycerophospholipid synthesis defects by modulating sphingolipid metabolism. Quantitative imaging further indicates that Ire1 mediates these effects, at least in part, by elevating cytoplasmic Ca2+ which in turn stimulates calcineurin activity necessary for cellular homeostasis. Accordingly, inhibition of calcineurin results in severe endoplasmic reticulum stress in yeast cells depleted of inter-organelle contacts. Thus, the Ire1 MSR directs Ca2+-dependent calcineurin activity and lipid metabolism upon disruptions in membrane contact sites to maintain cellular homeostasis. Alterations in inter-organelle contacts are associated with several diseases, including neurodegenerative disorders. Our findings in yeast suggest that evoking the MSR may be a means to ameliorate neuronal degeneration and delay the progression of neurodegenerative disorders.

cell biology↗

bioRxiv2021

Interactions of cytosolic termini of the Jen1 monocarboxylate transporter are critical for trafficking, transport activity and endocytosis

Plasma membrane (PM) transporters of the major facilitator superfamily (MFS) are essential for cell metabolism and growth, as well as for survival in response to stress or cytotoxic drugs, in both prokaryotes and eukaryotes. In the yeast Saccharomyces cerevisiae, Jen1 is a monocarboxylate/H+ symporter that has been used to dissect the molecular details underlying control of cellular expression, transport mechanism and turnover of MFS transporters. Here, we present evidence supporting previously non-described roles of the cytosolic N- and C- termini in Jen1 biogenesis, PM stability and activity, through functional analyses of rationally designed truncations and chimeric constructs with UapA, a S. cerevisiae endocytosis-insensitive purine transporter from Aspergillus nidulans. Our results reveal a cryptic role of the N-terminal region and thus show that both cytosolic N- and C-termini are critical for Jen1 trafficking to the PM, transport activity and endocytosis. In particular, we provide evidence that the N- and the C-cytosolic termini of Jen1 undergo transport-dependent dynamic intra-molecular interactions, which critically affect the mechanism of transport and turnover of Jen1. Our results support an emerging concept where the cytosolic tails of PM transporters control transporter expression and function, through flexible intra-molecular interactions with each other and the transmembrane core of the protein. This idea may be extended to other MFS members providing a deeper understanding of conserved, but also evolving, mechanisms underlying MFS transporter structure-function relationships.

microbiology↗

bioRxiv2021

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 endoplasmic reticulum and plasma membrane (ER-PM contacts). However, roles of the E-Syt family members in PM lipid organisation remain unclear. 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 promote the recruitment of Pkh1, a stress-activated protein kinase required for PM integrity. Phosphatidylserine has evolutionarily conserved roles in PM organisation, integrity, and repair. We suggest that phospholipid regulation is an ancient essential function of E-Syt family members in PM integrity.

cell biology↗

bioRxiv2020

ER-PM contacts regulate apical domain formation in hepatocytes

Apico-basal membrane polarity is fundamental for epithelial cell development and function. Polarity factors including the small GTPase Cdc42, the Par3/Par6/aPKC complex, and cytoskeletal proteins are recruited by the anionic lipids phosphatidylinositol 4,5-bisphosphate and phosphatidylserine. But how these lipids accumulate at polarised sites remains unclear. We have examined roles of contacts between the endoplasmic reticulum and plasma membrane (ER-PM contacts) in generating lipid gradients during apical domain formation. Comprehensive electron microscopy analyses in hepatocytes and epithelial spheroids revealed two distinct ER-PM contact architectures that are spatially linked to apical and baso-lateral domains. Moreover, apical domain formation was delayed in HepG2 cells upon modulating the ER-PM contact proteins E-Syt1 and ORP5. We propose ER-PM contacts regulate apico-basal polarity via the lipid transfer proteins E-Syt1 and ORP5. Importantly, our findings suggest that the spatial organisation of ER-PM contacts is a conserved feature of polarised epithelial cells.

cell biology↗

bioRxiv2020

Multiple roles for the ESCRT machinery in maintaining plasma membrane homeostasis

The endosomal sorting complexes required for transport (ESCRT) execute evolutionary conserved membrane remodeling processes. Here we used budding yeast to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. In response to reduced membrane tension and inhibition of the target of rapamycin complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help to maintain membrane integrity. Conversely, the growth of ESCRT mutants strongly depends on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This is caused by calcineurin phosphatase activity which causes Orm2 to accumulate at the endoplasmic reticulum (ER) in ESCRT mutants. Orm2 is a repressor of SL biosynthesis and its accumulation provokes increased membrane stress. This necessitates TORC2 signaling through its downstream kinase Ypk1 to control Orm2 protein levels and prevent a detrimental imbalance of SL metabolism. Our findings reveal new aspects of antagonistic calcineurin/TORC2 signaling for the regulation of SL biosynthesis and the maintenance of PM homeostasis, and suggest that the ESCRT machinery contributes directly and indirectly to these processes.

cell biology↗

bioRxiv2018

A Cytoplasmic GOLD Protein Controls Cell Polarity

Phosphoinositide lipids provide spatial landmarks during polarized secretion. Here, we elucidate a role for phosphatidylinositol 4-phosphate (PI4P) metabolism in the control of cell polarity. In budding yeast, PI4P is enriched at the plasma membrane of growing daughter cells. Upon heat shock however, PI4P rapidly increases at the plasma membrane in mother cells resulting in a more uniform PI4P distribution. Rather than phosphoinositide kinase activation, PI4P hydrolysis is impaired to generate the heat-induced PI4P signal in mother cells. This fine tune control of PI4P metabolism is mediated through attenuation of the Osh3 protein that binds and presents PI4P to a phosphoinositide phosphatase. Importantly, Osh3 undergoes phase transitions upon environmental stress conditions, resulting in intracellular aggregates and reduced cortical localization. The chaperone Hsp104 co-assembles with intracellular Osh3 granules, but is not required for their formation. Interestingly, the Osh3 GOLD domain, also present in the ER-localized p24 cargo adaptor family, is sufficient to form stress granules. Accordingly, GOLD-mediated phase transitions may provide a general mechanism to modulate secretion and growth upon transient changes in physiological and environmental conditions.

cell biology↗