scholarly journals Inner-Nuclear-Membrane-Associated Degradation Employs Dfm1-Independent Retrotranslocation and Alleviates Misfolded Transmembrane-Protein Toxicity

2021 ◽  
pp. mbc.E20-11-0720
Author(s):  
Matthew P. Flagg ◽  
Margaret A. Wangeline ◽  
Sarah R. Holland ◽  
Sascha H. Duttke ◽  
Christopher Benner ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Nuclear Membrane ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Extraction Process ◽  
26S Proteasome ◽  
Energetic Cost ◽  
Inner Nuclear Membrane ◽  
Transmembrane Segments ◽  
Protein Toxicity

Prior to their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the ER and inner-nuclear membrane must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the inner-nuclear membrane (INM), which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ERAD-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel, which can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.

scholarly journals Lysine 242 within Helix 10 of the Pseudorabies Virus Nuclear Egress Complex pUL31 Component Is Critical for Primary Envelopment of Nucleocapsids

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Sebastian Rönfeldt ◽  
Barbara G. Klupp ◽  
Kati Franzke ◽  
Thomas C. Mettenleiter
Keyword(s):  
Lipid Bilayers ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Amino Acid Position ◽  
Distal Portion ◽  
Lysine Residue ◽  
Interaction Domain ◽  
Inner Nuclear Membrane ◽  
Nuclear Egress ◽  
Membrane Budding

ABSTRACT Newly assembled herpesvirus nucleocapsids are translocated from the nucleus to the cytosol by a vesicle-mediated process engaging the nuclear membranes. This transport is governed by the conserved nuclear egress complex (NEC), consisting of the alphaherpesviral pUL34 and pUL31 homologs. The NEC is not only required for efficient nuclear egress but also sufficient for vesicle formation from the inner nuclear membrane (INM), as well as from synthetic lipid bilayers. The recently solved crystal structures for the NECs from different herpesviruses revealed molecular details of this membrane deformation and scission machinery uncovering the interfaces involved in complex and coat formation. However, the interaction domain with the nucleocapsid remained undefined. Since the NEC assembles a curved hexagonal coat on the nucleoplasmic side of the INM consisting of tightly interwoven pUL31/pUL34 heterodimers arranged in hexamers, only the membrane-distal end of the NEC formed by pUL31 residues appears to be accessible for interaction with the nucleocapsid cargo. To identify the amino acids involved in capsid incorporation, we mutated the corresponding regions in the alphaherpesvirus pseudorabies virus (PrV). Site-specifically mutated pUL31 homologs were tested for localization, interaction with pUL34, and complementation of PrV-ΔUL31. We identified a conserved lysine residue at amino acid position 242 in PrV pUL31 located in the alpha-helical domain H10 exposed on the membrane-distal end of the NEC as a key residue for nucleocapsid incorporation into the nascent primary particle. IMPORTANCE Vesicular transport through the nuclear envelope is a focus of research but is still not well understood. Herpesviruses pioneered this mechanism for translocation of the newly assembled nucleocapsid from the nucleus into the cytosol via vesicles derived from the inner nuclear membrane which fuse in a well-tuned process with the outer nuclear membrane to release their content. The structure of the viral nuclear membrane budding and scission machinery has been solved recently, providing in-depth molecular details. However, how cargo is incorporated remained unclear. We identified a conserved lysine residue in the membrane-distal portion of the nuclear egress complex required for capsid uptake into inner nuclear membrane-derived vesicles.

scholarly journals Membrane-anchored growth factor, HB-EGF, on the cell surface targeted to the inner nuclear membrane

2008 ◽  
Vol 180 (4) ◽  
pp. 763-769 ◽  
Author(s):  
Miki Hieda ◽  
Mayumi Isokane ◽  
Michiko Koizumi ◽  
Chiduru Higashi ◽  
Taro Tachibana ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Transmembrane Protein ◽  
Active Form ◽  
Type I ◽  
Transcriptional Repressors ◽  
Sequence Element ◽  
Inner Nuclear Membrane

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.

scholarly journals FAM209 associates with DPY19L2 and is required for sperm acrosome biogenesis and fertility in mice

2021 ◽  
Author(s):  
Julio M. Castaneda ◽  
Keisuke Shimada ◽  
Yuhkoh Satouh ◽  
Zhifeng Yu ◽  
Masahito Ikawa ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Proteomic Analysis ◽  
Transmembrane Protein ◽  
Sequence Similarity ◽  
Rare Condition ◽  
Mouse Sperm ◽  
Inner Nuclear Membrane ◽  
Infertile Men ◽  
Human And Mouse

Infertility afflicts up to 15% of couples globally each year with men a contributing factor in half of these cases. Globozoospermia is a rare condition found in infertile men that is characterized by defective acrosome biogenesis leading to the production of round shaped sperm. Here, we report a novel gene, Fam209 (Family with sequence similarity 209), that is required for acrosome biogenesis in mouse sperm. FAM209 is a small transmembrane protein conserved among mammals. Loss of Fam209 result in fertility defects secondary to abnormalities in acrosome biogenesis during spermiogenesis reminiscent of globozoospermia. Proteomic analysis of the FAM209 proteome identified DPY19L2, a protein involved in the majority of globozoospermia cases. While mutations in human and mouse DPY19L2 have been shown to cause globozoospermia, no in vivo interacting partners of DPY19L2 have been identified until now. FAM209 colocalizes with DPY19L2 to the inner nuclear membrane to maintain the developing acrosome. This report identifies FAM209 as the first interacting partner of DPY19L2 and the second protein that is essential for acrosome biogenesis and that co-localizes with DPY19L2 to the inner nuclear membrane.

Structural characterization of a C-terminally truncated E5 oncoprotein from papillomavirus in lipid bilayers

2014 ◽  
Vol 395 (12) ◽  
pp. 1443-1452 ◽  
Author(s):  
Dirk Windisch ◽  
Colin Ziegler ◽  
Jochen Bürck ◽  
Anne S. Ulrich
Keyword(s):  
Lipid Bilayers ◽  
Transmembrane Protein ◽  
Growth Factor Receptor ◽  
Helical Conformation ◽  
Resonance Spectroscopy ◽  
Bovine Papillomavirus ◽  
Transmembrane Segments ◽  
C Terminus ◽  
E5 Oncoprotein ◽  
Representative Model

AbstractE5 is the major transforming oncoprotein of bovine papillomavirus, which activates the platelet-derived growth factor receptor β in a highly specific manner. The short transmembrane protein E5 with only 44 residues interacts directly with the transmembrane segments of the receptor, but structural details are not available. Biophysical investigations are challenging, because the hydrophobic E5 protein tends to aggregate and get cross-linked non-specifically via two Cys residues near its C-terminus. Here, we demonstrate that a truncation by 10 amino acids creates a more manageable protein that can be conveniently used for structure analysis. Synchrotron radiation circular dichroism and solid-state15N- and31P-nuclear magnetic resonance spectroscopy show that this E5 variant serves as a representative model for the wild-type protein. The helical conformation of the transmembrane segment, its orientation in the lipid bilayer, and the ability to form homodimers in the membrane are not affected by the C-terminal truncation.

scholarly journals Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane

Nucleus ◽  
2016 ◽  
Vol 7 (4) ◽  
pp. 415-423 ◽  
Author(s):  
Balaje Vijayaraghavan ◽  
Mohammed Hakim Jafferali ◽  
Ricardo A. Figueroa ◽  
Einar Hallberg
Keyword(s):  
Nuclear Membrane ◽  
Transmembrane Protein ◽  
Inner Nuclear Membrane

scholarly journals Myne-1, a spectrin repeat transmembrane protein of the myocyte inner nuclear membrane, interacts with lamin A/C

2002 ◽  
Vol 115 (1) ◽  
pp. 61-70 ◽  
Author(s):  
John M. K. Mislow ◽  
Marian S. Kim ◽  
Dawn Belt Davis ◽  
Elizabeth M. McNally
Keyword(s):  
Muscular Dystrophy ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Lamin A ◽  
Spectrin Repeat ◽  
Inner Nuclear Membrane ◽  
C Terminus

Mutations in the genes encoding the inner nuclear membrane proteins lamin A/C and emerin produce cardiomyopathy and muscular dystrophy in humans and mice. The mechanism by which these broadly expressed gene products result in tissue-specific dysfunction is not known. We have identified a protein of the inner nuclear membrane that is highly expressed in striated and smooth muscle. This protein, myne-1 (myocyte nuclear envelope), is predicted to have seven spectrin repeats, an interrupted LEM domain and a single transmembrane domain at its C-terminus. We found that myne-1 is expressed upon early muscle differentiation in multiple intranuclear foci concomitant with lamin A/C expression. In mature muscle, myne-1 and lamin A/C are perfectly colocalized, although colocalization with emerin is only partial. Moreover, we show that myne-1 and lamin A/C coimmunoprecipitate from differentiated muscle in vitro. The muscle-specific inner nuclear envelope expression of myne-1, along with its interaction with lamin A/C, indicates that this gene is a potential mediator of cardiomyopathy and muscular dystrophy.

LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis

1998 ◽  
Vol 111 (10) ◽  
pp. 1441-1451 ◽  
Author(s):  
I. Duband-Goulet ◽  
J.C. Courvalin ◽  
B. Buendia
Keyword(s):  
Nuclear Membrane ◽  
Binding Protein ◽  
Transmembrane Protein ◽  
Chromatin Condensation ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain ◽  
Late Stages

Chromatin condensation and apposition to the nuclear envelope is an important feature of the execution phase of apoptosis. During this process, lamin proteins that are located between the inner nuclear membrane and heterochromatin are proteolyzed by the apoptosis-specific protease caspase 6. We have investigated the fate of nuclear membranes during apoptosis by studying the lamin B receptor (LBR), a transmembrane protein of the inner nuclear membrane. LBR interacts through its nucleoplasmic amino-terminal domain with both heterochromatin and B-type lamins, and is phosphorylated throughout the cell cycle, but on different sites in interphase and mitosis. We report here that: (i) the amino-terminal domain of LBR is specifically cleaved during apoptosis to generate an approximately 20 kDa soluble fragment; (ii) the cleavage of LBR is a late event of apoptosis and occurs subsequent to lamin B cleavage; (iii) the phosphorylation of LBR during apoptosis is similar to that occurring in interphase. As the association of condensed chromatin with the inner nuclear membrane persists until the late stages of apoptosis, we suggest that the chromatin binding protein LBR plays a major role in maintaining this association.

scholarly journals Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

2019 ◽  
Author(s):  
Wai Cheng (Christine) Wong ◽  
Jz-Yuan Juo ◽  
Yi-Hung Liao ◽  
Ching-Ya Cheng ◽  
Chih-Hsiang Lin ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Protein Dynamics ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Diffusion Coefficients ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Cell Plasma

AbstractNative cell membrane derived supported lipid bilayers (SLBs) are emerging platforms that have broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact of the bilayer membrane to the supporting substrate, and thus the mobility of transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins of distinct structures, i.e., GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescence proteins (GFPs) and their dynamics were measured by fluorescence single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length to protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1,000 to 5,000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were raised by increasing the polymer length, whereas the diffusion coefficient of GPI-anchored protein remained almost identical with different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm2/s approximately) in the cushioned membrane with the longest polymer length (molecular weight of 5,000), indicating that the SLBs were fully suspended from the substrate by the polymer cushion at the microscopic length scale. Transient confinements were observed from all three proteins, and increasing the polymer length reduced the tendency of transient confinements. The measured dynamics of membrane proteins were found to be nearly unchanged after depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer cushioned plasma membrane bilayers that are potentially useful for future developments of membrane-based biosensors and analytical assays.

scholarly journals Evolvement of LEM proteins as chromatin tethers at the nuclear periphery

2011 ◽  
Vol 39 (6) ◽  
pp. 1735-1741 ◽  
Author(s):  
Andreas Brachner ◽  
Roland Foisner
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Nuclear Periphery ◽  
Transmembrane Proteins ◽  
Signal Transducer ◽  
Protein Inhibitor ◽  
Inner Nuclear Membrane ◽  
Unicellular Eukaryotes ◽  
Attachment Factor ◽  
Affect Gene Expression

The nuclear envelope in eukaryotic cells has important roles in chromatin organization. The inner nuclear membrane contains over 60 transmembrane proteins. LEM [LAP2 (lamina-associated polypeptide 2)/emerin/MAN1] domain-containing proteins of the inner nuclear membrane are involved in tethering chromatin to the nuclear envelope and affect gene expression. They contain a common structural, bihelical motif, the so-called LEM domain, which mediates binding to a conserved chromatin protein, BAF (barrier to autointegration factor). Interestingly, this domain is highly related to other bihelical motifs, termed HeH (helix–extension–helix) and SAP {SAF (scaffold attachment factor)/acinus/PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)]} motifs, which are directly linked to DNA. In the present paper, we summarize evidence that the LEM motif evolved from the HeH and SAP domains concomitantly with BAF. In addition, we discuss the potential evolution of HeH/SAP and LEM domain-containing proteins and their role in chromatin tethering and gene regulation from unicellular eukaryotes to mammals.

Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

2020 ◽  
Vol 477 (14) ◽  
pp. 2715-2720
Author(s):  
Susana Castro-Obregón
Keyword(s):  
Cellular Senescence ◽  
Nuclear Membrane ◽  
Chromatin Structure ◽  
Cholesterol Synthesis ◽  
Reductase Activity ◽  
Chimeric Protein ◽  
Nuclear Lamina ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Lamin B Receptor

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

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