scholarly journals Dysferlin Domain-containing Proteins, Pex30p and Pex31p, Localized to Two Compartments, Control the Number and Size of Oleate-induced Peroxisomes in Pichia pastoris

2008 ◽  
Vol 19 (3) ◽  
pp. 885-898 ◽  
Author(s):  
Mingda Yan ◽  
Dorian A. Rachubinski ◽  
Saurabh Joshi ◽  
Richard A. Rachubinski ◽  
Suresh Subramani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Steady State ◽  
Pichia Pastoris ◽  
Subcellular Localization ◽  
Yarrowia Lipolytica ◽  
Dependent Manner ◽  
Growth Environment ◽  
Dual Localization

Yarrowia lipolytica Pex23p and Saccharomyces cerevisiae Pex30p, Pex31p, and Pex32p comprise a family of dysferlin domain–containing peroxins. We show that the deletion of their Pichia pastoris homologues, PEX30 and PEX31, does not affect the function or division of methanol-induced peroxisomes but results in fewer and enlarged, functional, oleate-induced peroxisomes. Synthesis of Pex30p is constitutive, whereas that of Pex31p is oleate-induced but at a much lower level relative to Pex30p. Pex30p interacts with Pex31p and is required for its stability. At steady state, both Pex30p and Pex31p exhibit a dual localization to the endoplasmic reticulum (ER) and peroxisomes. However, Pex30p is localized mostly to the ER, whereas Pex31p is predominantly on peroxisomes. Consistent with ER-to-peroxisome trafficking of these proteins, Pex30p accumulates on peroxisomes upon overexpression of Pex31p. Additionally, Pex31p colocalizes with Pex30p at the ER in pex19Δ cells and can be chased from the ER to peroxisomes in a Pex19p-dependent manner. The dysferlin domains of Pex30p and Pex31p, which are dispensable for their interaction, stability, and subcellular localization, are essential for normal peroxisome number and size. The growth environment-specific role of these peroxins, their dual localization, and the function of their dysferlin domains provide novel insights into peroxisome morphogenesis.

scholarly journals Phosphorylation-dependent Pex11p and Fis1p interaction regulates peroxisome division

2012 ◽  
Vol 23 (7) ◽  
pp. 1307-1315 ◽  
Author(s):  
Saurabh Joshi ◽  
Gaurav Agrawal ◽  
Suresh Subramani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Pichia Pastoris ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Protein Levels ◽  
Protein Biogenesis ◽  
Peroxisome Division ◽  
Dependent Interaction

Peroxisome division is regulated by the conserved peroxin Pex11p. In Saccharomyces cerevisiae (Sc), induction of the phosphoprotein ScPex11p coincides with peroxisome biogenesis. We show that the ScPex11p homologue in Pichia pastoris (PpPex11p) is phosphorylated at serine 173. PpPex11p expression and phosphorylation are induced in oleate and coordinated with peroxisome biogenesis. PpPex11p transits to peroxisomes via the endoplasmic reticulum (ER). PpPex11p is unstable and ER restricted gin pex3Δ and pex19Δ cells, which are impaired in peroxisomal membrane protein biogenesis. In oleate medium, the P. pastoris mutants pex11A (constitutively unphosphorylated; S173A) and pex11D (constitutively phosphorylated; S173D) exhibit juxtaposed elongated peroxisomes (JEPs) and hyperdivided forms, respectively, although protein levels remain unchanged. In contrast with ScPex11p, the ER-to-peroxisome translocation in P. pastoris is phosphorylation independent, and the phosphorylation occurs at the peroxisome. We show that PpPex11p interacts with the peroxisome fission machinery via PpFis1p and is regulated by phosphorylation because PpPex11p and PpPex11Dp interact more strongly with PpFis1p than PpPex11Ap. Neither PpPex11p nor PpFis1p is necessary for peroxisome division in methanol medium. We propose a model for the role of PpPex11p in the regulation of peroxisome division through a phosphorylation-dependent interaction with the fission machinery, providing novel insights into peroxisome morphogenesis.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

Uptake and trafficking of exogenous sterols in Saccharomyces cerevisiae

2006 ◽  
Vol 34 (3) ◽  
pp. 359-362 ◽  
Author(s):  
S. Raychaudhuri ◽  
W.A. Prinz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Abc Transporters ◽  
Cellular Functions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Membrane Function ◽  
Oxysterol Binding Protein ◽  
Atp Binding Cassette Transporters ◽  
Related Proteins

The proper distribution of sterols among organelles is critical for numerous cellular functions. How sterols are sorted and moved among membranes remains poorly understood, but they are transported not only in vesicles but also by non-vesicular pathways. One of these pathways moves exogenous sterols from the plasma membrane to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae. We have found that two classes of proteins play critical roles in this transport, ABC transporters (ATP-binding-cassette transporters) and oxysterol-binding protein-related proteins. Transport is also regulated by phosphoinositides and the interactions of sterols with other lipids. Here, we summarize these findings and speculate on the role of non-vesicular sterol transfer in determining intracellular sterol distribution and membrane function.

scholarly journals Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

1996 ◽  
Vol 7 (5) ◽  
pp. 769-789 ◽  
Author(s):  
A J Koning ◽  
C J Roberts ◽  
R L Wright
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Subcellular Localization ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Endoplasmic Reticulum Membrane ◽  
Hmg Coa Reductase ◽  
Endogenous Expression ◽  
Coa Reductase ◽  
Er Membrane

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.

The role of endoplasmic reticulum stress in lead (Pb)-induced mitophagy of HEK293 cells

2020 ◽  
Vol 36 (12) ◽  
pp. 1002-1009
Author(s):  
Ke Gao ◽  
Chengfei Zhang ◽  
Yihong Tian ◽  
Sajid Naeem ◽  
Yingmei Zhang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Damage ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Pb Toxicity ◽  
Hsp60 Expression ◽  
Living Organisms ◽  
Almost All

It is well-documented that lead (Pb) toxicity can affect almost all systems in living organisms. It can induce selective autophagy of mitochondria (mitophagy) by triggering reactive oxygen species production. Emerging evidence has suggested that Pb-induced autophagy can also be activated by the endoplasmic reticulum (ER) stress pathway. However, the interplay between ER stress and mitophagy remains to be elucidated. In this study, human embryonic kidney HEK293 cells were employed to investigate the role of ER stress in Pb-induced mitophagy. The results showed that the cell viability was decreased and cell damage was induced after exposure to Pb (0, 0.5, 1, 2, and 4 mM) for 24 h in a dose-dependent manner. Moreover, the expression of LC3-Ⅱ was significantly increased, and the expression of HSP60 was dramatically decreased after exposure to 1 mM and 2 mM Pb, indicating the induction of mitophagy following Pb exposure. Meanwhile, the expressions of activating transcription factor 6, inositol-requiring protein-1α, CCAAT/enhancer binding protein homologous protein, and glucose-regulated protein 78 were dramatically increased after Pb treatment, signifying the initiation of ER stress. Notably, the mitophagic effect was significantly compromised when ER stress was inhibited by 0.5 mM 4-phenylbutyrate, which was evidenced by lesser decreases in HSP60 expression and level of LC3-Ⅱ, suggesting Pb-induced mitophagy may be activated by the ER stress. Taken together, these findings provide a better understanding of Pb toxicity and suggest that Pb-induced ER stress may play a regulatory role in the upstream of mitophagy.

scholarly journals mRNAs Encoding Polarity and Exocytosis Factors Are Cotransported with the Cortical Endoplasmic Reticulum to the Incipient Bud in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (9) ◽  
pp. 3441-3455 ◽  
Author(s):  
Stella Aronov ◽  
Rita Gelin-Licht ◽  
Gadi Zipor ◽  
Liora Haim ◽  
Einat Safran ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Nuclear Division ◽  
Mrna Localization ◽  
Dependent Manner ◽  
Protein Enrichment ◽  
Bud Emergence ◽  
Bud Site ◽  
Cortical Endoplasmic Reticulum ◽  
Asymmetrically Distributed

ABSTRACT Polarized growth in the budding yeast Saccharomyces cerevisiae depends upon the asymmetric localization and enrichment of polarity and secretion factors at the membrane prior to budding. We examined how these factors (i.e., Cdc42, Sec4, and Sro7) reach the bud site and found that their respective mRNAs localize to the tip of the incipient bud prior to nuclear division. Asymmetric mRNA localization depends upon factors that facilitate ASH1 mRNA localization (e.g., the 3′ untranslated region, She proteins 1 to 5, Puf6, actin cytoskeleton, and a physical association with She2). mRNA placement precedes protein enrichment and subsequent bud emergence, implying that mRNA localization contributes to polarization. Correspondingly, mRNAs encoding proteins which are not asymmetrically distributed (i.e., Snc1, Mso1, Tub1, Pex3, and Oxa1) are not polarized. Finally, mutations which affect cortical endoplasmic reticulum (ER) entry and anchoring in the bud (myo4Δ, sec3Δ, and srp101) also affect asymmetric mRNA localization. Bud-localized mRNAs, including ASH1, were found to cofractionate with ER microsomes in a She2- and Sec3-dependent manner; thus, asymmetric mRNA transport and cortical ER inheritance are connected processes in yeast.

ПЕРСПЕКТИВЫ ИСПОЛЬЗОВАНИЯ ДРОЖЖЕВОГО ДИСПЛЕЯ В?БИОТЕХНОЛОГИИ И КЛЕТОЧНОЙ БИОЛОГИИ (ОБЗОР), "Прикладная биохимия и микробиология"

2018 ◽  
pp. 329-346
Author(s):  
М.В. Падкина ◽  
Е.В. Самбук
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Yarrowia Lipolytica

Технология дрожжевого дисплея, появившаяся 20 лет назад и основанная на экспонировании на поверхности клетки целевых белков за счет их слияния с белками клеточной стенки, находит все более широкое применение в фундаментальных и прикладных исследованиях. К основному преимуществу поверхностного дисплея на основе эукариотических микроорганизмов - дрожжей относится возможность корректной модификации белков млекопитающих. Поверхностный дисплей является одним из инструментов изучения функций белков, межбелковых взаимодействий, анализа и селекции пептидных и белковых библиотек. Данная технология позволяет получать клетки дрожжей, которые обладают такими новыми свойствами, как каталитическая активность, способность связывать определенные лиганды, в том числе, редкие и тяжелые металлы. Это дает возможность использовать дрожжи в биотехнологии, а также для биоремедиации и биомониторинга окружающей среды. В обзоре рассмотрены способы получения поверхностного дисплея на основе дрожжей Saccharomyces cerevisiae, Pichia pastoris и Yarrowia lipolytica, свойства якорных белков и основные области применения технологии дрожжевого дисплея.

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

2020 ◽  
Vol 133 (21) ◽  
pp. jcs248526 ◽  
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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