scholarly journals FIT2 is a lipid phosphate phosphatase crucial for endoplasmic reticulum homeostasis

2018 ◽  
Author(s):  
Michel Becuwe ◽  
Laura M. Bond ◽  
Niklas Mejhert ◽  
Sebastian Boland ◽  
Shane D. Elliott ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Morphological Changes ◽  
Normal Structure ◽  
Membrane Lipid ◽  
Molecular Function ◽  
Key Factor ◽  
Lipid Phosphate ◽  
Lipid Abnormalities ◽  
Er Membrane

SUMMARYThe endoplasmic reticulum (ER) protein Fat-Induced Transcript 2 (FIT2) has emerged as a key factor in lipid droplet (LD) formation, although its molecular function is unknown. Highlighting its importance, FIT2 orthologs are essential in worms and mice, and FIT2 deficiency causes a deafness/dystonia syndrome in humans. Here we show that FIT2 is a lipid phosphate phosphatase (LPP) enzyme that is required for maintaining the normal structure of the ER. Recombinant FIT2 exhibits LPP activityin vitroand loss of this activity in cells leads to ER membrane morphological changes and ER stress. Defects in LD formation in FIT2 depletion appear to be secondary to membrane lipid abnormalities, possibly due to alterations in phospholipids required for coating forming LDs. Our findings uncover an enzymatic role for FIT2 in ER lipid metabolism that is crucial for ER membrane homeostasis.

scholarly journals TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (8) ◽  
pp. 2231-2247 ◽  
Author(s):  
Julia D. Romano ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Eukaryotic Proteins ◽  
Internal Site ◽  
Membrane Spanning ◽  
Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

scholarly journals Membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor Ire1 in different ways

2011 ◽  
Vol 22 (18) ◽  
pp. 3520-3532 ◽  
Author(s):  
Thanyarat Promlek ◽  
Yuki Ishiwata-Kimata ◽  
Masahiro Shido ◽  
Mitsuru Sakuramoto ◽  
Kenji Kohno ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Membrane Lipid ◽  
Yeast Cells ◽  
Wild Type ◽  
Unfolded Proteins ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Eukaryotic cells activate the unfolded-protein response (UPR) upon endoplasmic reticulum (ER) stress, where the stress is assumed to be the accumulation of unfolded proteins in the ER. Consistent with previous in vitro studies of the ER-luminal domain of the mutant UPR initiator Ire1, our study show its association with a model unfolded protein in yeast cells. An Ire1 luminal domain mutation that compromises Ire1's unfolded-protein–associating ability weakens its ability to respond to stress stimuli, likely resulting in the accumulation of unfolded proteins in the ER. In contrast, this mutant was activated like wild-type Ire1 by depletion of the membrane lipid component inositol or by deletion of genes involved in lipid homeostasis. Another Ire1 mutant lacking the authentic luminal domain was up-regulated by inositol depletion as strongly as wild-type Ire1. We therefore conclude that the cytosolic (or transmembrane) domain of Ire1 senses membrane aberrancy, while, as proposed previously, unfolded proteins accumulating in the ER interact with and activate Ire1.

Topological and Enzymatic Analysis of Human Alg2 Mannosyltransferase

2021 ◽  
Author(s):  
Meng-Hai Xiang ◽  
Xin-Xin Xu ◽  
Chun-Di Wang ◽  
Shuai Chen ◽  
Si Xu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Biosynthetic Pathway ◽  
Membrane Binding ◽  
Binding Domain ◽  
Enzymatic Analysis ◽  
Kinetic Assay ◽  
Physiological Conditions ◽  
Single Membrane ◽  
Er Membrane

Abstract N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum. Alg2 mannosyltransferase adds both the α1,3- and α1,6-Man onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged significantly between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop an LC-MS quantitative kinetic assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man on to M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2 (α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.

scholarly journals Vulnerability of Subcellular Structures to Pathogenesis Induced by Rotenone in SH-SY5Y Cells

2021 ◽  
pp. 89-99
Author(s):  
M POKUSA ◽  
D HAJDÚCHOVÁ ◽  
V MENICHOVÁ ◽  
A EVINOVÁ ◽  
Z HATOKOVÁ ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Morphological Changes ◽  
Protein Accumulation ◽  
Main Research ◽  
Microtubular Cytoskeleton ◽  
Intracellular Compartments ◽  
Biochemical Analyses ◽  
Microtubular System

Numerous pathological changes of subcellular structures are characteristic hallmarks of neurodegeneration. The main research has focused to mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomal networks as well as microtubular system of the cell. The sequence of specific organelle damage during pathogenesis has not been answered yet. Exposition to rotenone is used for simulation of neurodegenerative changes in SH-SY5Y cells, which are widely used for in vitro modelling of Parkinson´s disease pathogenesis. Intracellular effects were investigated in time points from 0 to 24 h by confocal microscopy and biochemical analyses. Analysis of fluorescent images identified the sensitivity of organelles towards rotenone in this order: microtubular cytoskeleton, mitochondrial network, endoplasmic reticulum, Golgi apparatus and lysosomal network. All observed morphological changes of intracellular compartments were identified before αS protein accumulation. Therefore, their potential as an early diagnostic marker is of interest. Understanding of subcellular sensitivity in initial stages of neurodegeneration is crucial for designing new approaches and a management of neurodegenerative disorders.

scholarly journals Membrane integration of Sec61α: a core component of the endoplasmic reticulum translocation complex

1998 ◽  
Vol 331 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Bruce C. KNIGHT ◽  
Stephen HIGH
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Insertion Site ◽  
Secretory Protein ◽  
Cross Linking ◽  
Central Component ◽  
Membrane Insertion ◽  
Er Membrane

The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61α, Sec61β and Sec61γ, at least two of which (α and β) are adjacent to nascent proteins during membrane insertion. Another component of the translocation machinery is the translocating chain-associating membrane (TRAM) protein, which is also adjacent to many nascent proteins during membrane insertion. Sec61α functions as the major component of a transmembrane channel formed by oligomers of the Sec61 complex. This channel is the site of secretory protein translocation and membrane protein integration at the ER membrane. Sec61α is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a cross-linking approach to analyse the environment of a series of discrete Sec61α membrane-integration intermediates, we find: (i) newly synthesized Sec61α is adjacent to known components of the ER membrane-insertion site, namely Sec61α, Sec61β and TRAM, and thus the integration of Sec61α appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61α remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61β) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61α requires ER targeting by the signal-recognition particle.

scholarly journals Carrier-mediated transport of uridine diphosphoglucuronic acid across the endoplasmic reticulum membrane is a prerequisite for UDP-glucuronosyltransferase activity in rat liver

1997 ◽  
Vol 323 (3) ◽  
pp. 645-648 ◽  
Author(s):  
Xavier BOSSUYT ◽  
Norbert BLANCKAERT
Keyword(s):  
Endoplasmic Reticulum ◽  
Permeability Barrier ◽  
Endoplasmic Reticulum Membrane ◽  
Carrier Mediated Transport ◽  
Rate Limiting ◽  
Isolated Liver ◽  
Stimulation Of ◽  
Er Membrane

UDP-glucuronosyltransferases (EC 2.4.1.17) is an isoenzyme family located primarily in the hepatic endoplasmic reticulum (ER) that displays latency of activity both in vitro and in vivo, as assessed respectively in microsomes and in isolated liver. The postulated luminal location of the active site of UDP-glucuronosyltransferases (UGTs) creates a permeability barrier to aglycone and UDP-GlcA access to the enzyme and implies a requirement for the transport of substrates across the ER membrane. The present study shows that the recently demonstrated carrier-mediated transport of UDP-GlcA across the ER membrane is required and rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes. We found that in both microsomes and permeabilized hepatocytes a gradual inhibition by N-ethylmaleimide (NEM) of UDP-GlcA transport into the ER produced a correspondingly increasing inhibition of 4-methylumbelliferone glucuronidation. That NEM selectively inhibited the UDP-GlcA transporter, without affecting intrinsic UGT activity, was demonstrated by showing that NEM had no effect on glucuronidation in microsomes or hepatocytes with permeabilized ER membrane. Additional evidence that UDP-GlcA transport is rate-limiting for glucuronidation in sealed microsomal vesicles as well as in the intact ER of permeabilized hepatocytes was obtained by showing that gradual selective trans-stimulation of UDP-GlcA transport by UDP-GlcNAc, UDP-Xyl or UDP-Glc in each case produced correspondingly enhanced glucuronidation. Such stimulation of transport and glucuronidation was inhibited completely by NEM, which selectively inhibited UDP-GlcA transport.

scholarly journals Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci

2005 ◽  
Vol 86 (5) ◽  
pp. 1415-1421 ◽  
Author(s):  
Sarah N. Gretton ◽  
Annette I. Taylor ◽  
John McLauchlan
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Morphological Changes ◽  
Hcv Rna ◽  
Endoplasmic Reticulum Membrane ◽  
Structural Protein ◽  
Direct Role ◽  
Cellular Marker ◽  
Er Membrane

The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP–NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These membrane-associated foci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.

scholarly journals Metabolic activity induces membrane phase separation in endoplasmic reticulum

2017 ◽  
Vol 114 (51) ◽  
pp. 13394-13399 ◽  
Author(s):  
Yihui Shen ◽  
Zhilun Zhao ◽  
Luyuan Zhang ◽  
Lingyan Shi ◽  
Sanjid Shahriar ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Phase Behavior ◽  
Metabolic Activity ◽  
Unsaturated Fatty Acids ◽  
Solid Phase ◽  
Cellular Membrane ◽  
Membrane Phase ◽  
Er Membrane

Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.

scholarly journals Morphological Heterogeneity of the Endoplasmic Reticulum within Neurons and Its Implications in Neurodegeneration

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 970
Author(s):  
Sreesha Sree ◽  
Ilmari Parkkinen ◽  
Anna Their ◽  
Mikko Airavaara ◽  
Eija Jokitalo
Keyword(s):  
Endoplasmic Reticulum ◽  
Neurodegenerative Diseases ◽  
Dopaminergic Neurons ◽  
Morphological Changes ◽  
Lipid Homeostasis ◽  
Morphological Attributes ◽  
Morphological Heterogeneity ◽  
Neuronal Types ◽  
Er Membrane

The endoplasmic reticulum (ER) is a multipurpose organelle comprising dynamic structural subdomains, such as ER sheets and tubules, serving to maintain protein, calcium, and lipid homeostasis. In neurons, the single ER is compartmentalized with a careful segregation of the structural subdomains in somatic and neurite (axodendritic) regions. The distribution and arrangement of these ER subdomains varies between different neuronal types. Mutations in ER membrane shaping proteins and morphological changes in the ER are associated with various neurodegenerative diseases implying significance of ER morphology in maintaining neuronal integrity. Specific neurons, such as the highly arborized dopaminergic neurons, are prone to stress and neurodegeneration. Differences in morphology and functionality of ER between the neurons may account for their varied sensitivity to stress and neurodegenerative changes. In this review, we explore the neuronal ER and discuss its distinct morphological attributes and specific functions. We hypothesize that morphological heterogeneity of the ER in neurons is an important factor that accounts for their selective susceptibility to neurodegeneration.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

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