Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae

Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes.

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Activation of the Mitogen-activated Protein Kinase, Slt2p, at Bud Tips Blocks a Late Stage of Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e09-06-0532 ◽

2010 ◽

Vol 21 (10) ◽

pp. 1772-1782 ◽

Cited By ~ 13

Author(s):

Xia Li ◽

Yunrui Du ◽

Steven Siegel ◽

Susan Ferro-Novick ◽

Peter Novick

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Protein Phosphatase ◽

Normal Growth ◽

Growth Conditions ◽

Mitogen Activated Protein Kinase ◽

Mitochondrial Inheritance ◽

Daughter Cells ◽

Late Step ◽

Mitogen Activated Protein

Inheritance of the endoplasmic reticulum (ER) requires Ptc1p, a type 2C protein phosphatase of Saccharomyces cerevisiae . Genetic analysis indicates that Ptc1p is needed to inactivate the cell wall integrity (CWI) MAP kinase, Slt2p. Here we show that under normal growth conditions, Ptc1p inactivates Slt2p just as ER tubules begin to spread from the bud tip along the cortex. In ptc1Δ cells, the propagation of cortical ER from the bud tip to the periphery of the bud is delayed by hyperactivation of Slt2p. The pool of Slt2p that controls ER inheritance requires the CWI pathway scaffold, Spa2p, for its retention at the bud tip, and a mutation within Slt2p that prevents its association with the bud tip blocks its role in ER inheritance. These results imply that Slt2p inhibits a late step in ER inheritance by phosphorylating a target at the tip of daughter cells. The PI4P5-kinase, Mss4p, is an upstream activator of this pool of Slt2p. Ptc1p-dependant inactivation of Slt2p is also needed for mitochondrial inheritance; however, in this case, the relevant pool of Slt2p is not at the bud tip.

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Compartmentalized Reconstitution of Post-squalene Pathway for 7-Dehydrocholesterol Overproduction in Saccharomyces cerevisiae

Frontiers in Microbiology ◽

10.3389/fmicb.2021.663973 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiao-Jing Guo ◽

Ming-Dong Yao ◽

Wen-Hai Xiao ◽

Ying Wang ◽

Guang-Rong Zhao ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Vitamin D ◽

Endoplasmic Reticulum ◽

Shake Flask ◽

Metabolic Flux ◽

Heterologous Protein ◽

Subcellular Location ◽

Product Yield ◽

Lipid Bodies ◽

Level Production

7-Dehydrocholesterol (7-DHC) is the direct precursor to manufacture vitamin D3. Our previous study has achieved 7-DHC synthesis in Saccharomyces cerevisiae based on the endogenous post-squalene pathway. However, the distribution of post-squalene enzymes between the endoplasmic reticulum (ER) and lipid bodies (LD) might raise difficulties for ERG proteins to catalyze and deliver sterol intermediates, resulting in unbalanced metabolic flow and low product yield. Herein, we intended to rearrange the subcellular location of post-squalene enzymes to alleviate metabolic bottleneck and boost 7-DHC production. After identifying the location of DHCR24 (C-24 reductase, the only heterologous protein for 7-DHC biosynthesis) on ER, all the ER-located enzymes were grouped into four modules: ERG1/11/24, ERG25/26/27, ERG2/3, and DHCR24. These modules attempted to be overexpressed either on ER or on LDs. As a result, expression of LD-targeted DHCR24 and ER-located ERG1/11/24 could promote the conversion efficiency among the sterol intermediates to 7-DHC, while locating module ERG2/3 into LDs improved the whole metabolic flux of the post-squalene pathway. Coexpressing LD-targeted ERG2/3 and DHCR24 (generating strain SyBE_Sc01250035) improved 7-DHC production from 187.7 to 308.2 mg/L at shake-flask level. Further expressing ER-targeted module ERG1/11/24 in strain SyBE_Sc01250035 dramatically reduced squalene accumulation from 620.2 mg/L to the lowest level (by 93.8%) as well as improved 7-DHC production to the highest level (to 342.2 mg/L). Then targeting module ERG25/26/27 to LDs further increased 7-DHC titer to 360.6 mg/L, which is the highest shake-flask level production for 7-DHC ever reported. Our study not only proposes and further proves the concept of pathway compartmentalized reconstitution to regulate metabolic flux but also provides a promising chassis to produce other steroidal compounds through the post-squalene pathway.

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The yeast FIT2 homologs are necessary to maintain cellular proteostasis and membrane lipid homeostasis

Journal of Cell Science ◽

10.1242/jcs.248526 ◽

2020 ◽

Vol 133 (21) ◽

pp. jcs248526 ◽

Cited By ~ 1

Author(s):

Wei Sheng Yap ◽

Peter Shyu ◽

Maria Laura Gaspar ◽

Stephen A. Jesch ◽

Charlie Marvalim ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Lipid Droplets ◽

Membrane Lipid ◽

Model System ◽

Lipid Homeostasis ◽

Compensatory Mechanism ◽

Unfolded Protein ◽

Protein Response

ABSTRACTLipid droplets (LDs) are implicated in conditions of lipid and protein dysregulation. The fat storage-inducing transmembrane (FIT; also known as FITM) family induces LD formation. Here, we establish a model system to study the role of the Saccharomyces cerevisiae FIT homologues (ScFIT), SCS3 and YFT2, in the proteostasis and stress response pathways. While LD biogenesis and basal endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) remain unaltered in ScFIT mutants, SCS3 was found to be essential for proper stress-induced UPR activation and for viability in the absence of the sole yeast UPR transducer IRE1. Owing to not having a functional UPR, cells with mutated SCS3 exhibited an accumulation of triacylglycerol within the ER along with aberrant LD morphology, suggesting that there is a UPR-dependent compensatory mechanism that acts to mitigate lack of SCS3. Additionally, SCS3 was necessary to maintain phospholipid homeostasis. Strikingly, global protein ubiquitylation and the turnover of both ER and cytoplasmic misfolded proteins is impaired in ScFITΔ cells, while a screen for interacting partners of Scs3 identifies components of the proteostatic machinery as putative targets. Together, our data support a model where ScFITs play an important role in lipid metabolism and proteostasis beyond their defined roles in LD biogenesis.This article has an associated First Person interview with the first author of the paper.

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Remodeling of secretory compartments creates CUPS during nutrient starvation

The Journal of Cell Biology ◽

10.1083/jcb.201407119 ◽

2014 ◽

Vol 207 (6) ◽

pp. 695-703 ◽

Cited By ~ 32

Author(s):

David Cruz-Garcia ◽

Amy J. Curwin ◽

Jean-François Popoff ◽

Caroline Bruns ◽

Juan M. Duran ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Coat Protein ◽

Nitrogen Starvation ◽

Growth Conditions ◽

Vesicle Tethering ◽

Copii Vesicle ◽

Peripheral Membrane Protein ◽

Er Exit Sites ◽

Endosomal Membranes

Upon starvation, Grh1, a peripheral membrane protein located at endoplasmic reticulum (ER) exit sites and early Golgi in Saccharomyces cerevisiae under growth conditions, relocates to a compartment called compartment for unconventional protein secretion (CUPS). Here we report that CUPS lack Golgi enzymes, but contain the coat protein complex II (COPII) vesicle tethering protein Uso1 and the Golgi t-SNARE Sed5. Interestingly, CUPS biogenesis is independent of COPII- and COPI-mediated membrane transport. Pik1- and Sec7-mediated membrane export from the late Golgi is required for complete assembly of CUPS, and Vps34 is needed for their maintenance. CUPS formation is triggered by glucose, but not nitrogen starvation. Moreover, upon return to growth conditions, CUPS are absorbed into the ER, and not the vacuole. Altogether our findings indicate that CUPS are not specialized autophagosomes as suggested previously. We suggest that starvation triggers relocation of secretory and endosomal membranes, but not their enzymes, to generate CUPS to sort and secrete proteins that do not enter, or are not processed by enzymes of the ER–Golgi pathway of secretion.

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Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Biological Chemistry ◽

10.1515/bc.2000.126 ◽

2000 ◽

Vol 381 (9-10) ◽

pp. 1025-1028 ◽

Cited By ~ 23

Author(s):

Anke Prinz ◽

Enno Hartmann ◽

Kai-Uwe Kalies

Keyword(s):

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Binding Sites ◽

Mammalian Cells ◽

Protein Translocation ◽

Growth Conditions ◽

Ribosome Binding ◽

Ribosome Binding Sites ◽

Tight Association ◽

Biochemical Analyses

Abstract A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.

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Role of Tos3, a Snf1 Protein Kinase Kinase, during Growth of Saccharomyces cerevisiae on Nonfermentable Carbon Sources

Eukaryotic Cell ◽

10.1128/ec.4.5.861-866.2005 ◽

2005 ◽

Vol 4 (5) ◽

pp. 861-866 ◽

Cited By ~ 18

Author(s):

Myoung-Dong Kim ◽

Seung-Pyo Hong ◽

Marian Carlson

Keyword(s):

Saccharomyces Cerevisiae ◽

Catalytic Activity ◽

Protein Kinase ◽

Carbon Source ◽

Carbon Sources ◽

Growth Conditions ◽

Single Mutation ◽

Glucose Depletion ◽

Nonfermentable Carbon Source ◽

Responsive Element

ABSTRACT In Saccharomyces cerevisiae, Snf1 protein kinase of the Snf1/AMP-activated protein kinase family is required for growth on nonfermentable carbon sources and nonpreferred sugars. Three kinases, Pak1, Elm1, and Tos3, activate Snf1 by phosphorylation of its activation-loop threonine, and the absence of all three causes the Snf− phenotype. No phenotype has previously been reported for the tos3Δ single mutation. We show here that, when cells are grown on glycerol-ethanol, tos3Δ reduces growth rate, Snf1 catalytic activity, and activation of the Snf1-dependent carbon source-responsive element (CSRE) in the promoters of gluconeogenic genes. In contrast, tos3Δ did not significantly affect Snf1 catalytic activity or CSRE function during abrupt glucose depletion, indicating that Tos3 has a more substantial role in activating Snf1 protein kinase during growth on a nonfermentable carbon source than during acute carbon stress. We also report that Tos3 is localized in the cytosol during growth in either glucose or glycerol-ethanol. These findings lend support to the idea that the Snf1 protein kinase kinases make different contributions to cellular regulation under different growth conditions.

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