Detection of a Novel 9-kDa Endoplasmic Reticulum Membrane Protein in Mammalian Cells by Chemical Cross-Linking with Translocating Nascent Peptides1

1993 ◽  
Vol 114 (4) ◽  
pp. 541-546 ◽  
Author(s):  
Tomoko Kuroiwa ◽  
Masao Sakaguchi ◽  
Katsuyoshi Mihara ◽  
Tsuneo Omura
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Cross Linking ◽  
Endoplasmic Reticulum Membrane ◽  
Chemical Cross Linking

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

scholarly journals p180 Is Involved in the Interaction between the Endoplasmic Reticulum and Microtubules through a Novel Microtubule-binding and Bundling Domain

2007 ◽  
Vol 18 (10) ◽  
pp. 3741-3751 ◽  
Author(s):  
Kiyoko Ogawa-Goto ◽  
Keiko Tanaka ◽  
Tomonori Ueno ◽  
Keisuke Tanaka ◽  
Takeshi Kurata ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Specific Interaction ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Animal Cells ◽  
Microtubule Binding

p180 was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum membrane, although its precise role in animal cells has not yet been elucidated. Here, we characterized a new function of human p180 as a microtubule-binding and -modulating protein. Overexpression of p180 in mammalian cells induced an elongated morphology and enhanced acetylated microtubules. Consistently, electron microscopic analysis clearly revealed microtubule bundles in p180-overexpressing cells. Targeted depletion of endogenous p180 by small interfering RNAs led to aberrant patterns of microtubules and endoplasmic reticulum in mammalian cells, suggesting a specific interaction between p180 and microtubules. In vitro sedimentation assays using recombinant polypeptides revealed that p180 bound to microtubules directly and possessed a novel microtubule-binding domain (designated MTB-1). MTB-1 consists of a predicted coiled-coil region and repeat domain, and strongly promoted bundle formation both in vitro and in vivo when expressed alone. Overexpression of p180 induced acetylated microtubules in cultured cells in an MTB-1-dependent manner. Thus, our data suggest that p180 mediates interactions between the endoplasmic reticulum and microtubules mainly through the novel microtubule-binding and -bundling domain MTB-1.

scholarly journals Interactions between Sec Complex and Prepro-α-Factor during Posttranslational Protein Transport into the Endoplasmic Reticulum

2004 ◽  
Vol 15 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Kathrin Plath ◽  
Barrie M. Wilkinson ◽  
Colin J. Stirling ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Transport ◽  
Polypeptide Chain ◽  
Signal Sequence ◽  
Cross Linking ◽  
Endoplasmic Reticulum Membrane ◽  
Transmembrane Segments ◽  
Sequence Recognition ◽  
Multiple Regions ◽  
A Site

Posttranslational translocation of prepro-α-factor (ppαF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex. We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step. We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules. To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppαF was generated with photoreactive probes incorporated into the mature portion of the polypeptide. Cross-linking to multiple regions of Sec61p was observed. In contrast to the signal sequence, neighboring positions of the mature portion of ppαF had similar interactions with Sec61p. These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.

Grp78, Grp94, and Grp170 interact with α1-antitrypsin mutants that are retained in the endoplasmic reticulum

2005 ◽  
Vol 289 (3) ◽  
pp. G444-G455 ◽  
Author(s):  
Bela Z. Schmidt ◽  
David H. Perlmutter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cellular Response ◽  
Gradient Centrifugation ◽  
Calcium Ionophore ◽  
Genetically Engineered ◽  
Cross Linking ◽  
Ionophore A23187 ◽  
Mutant Form ◽  
Sucrose Density Gradient Centrifugation ◽  
Chemical Cross Linking

In α1-antitrypsin (α1-AT) deficiency, a mutant form of α1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant α1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The α1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which α1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant α1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of ∼110–130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic α1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the α1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that ∼85% of α1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and ∼15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of α1-AT Z as well as on its residual secretion.

scholarly journals Complexes between nascent polypeptides and their molecular chaperones in the cytosol of mammalian cells.

1997 ◽  
Vol 8 (8) ◽  
pp. 1559-1573 ◽  
Author(s):  
D K Eggers ◽  
W J Welch ◽  
W J Hansen
Keyword(s):  
Molecular Chaperones ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Cooperative Interaction ◽  
Cross Linking ◽  
New Approach ◽  
Physiological Conditions ◽  
Before And After ◽  
Chemical Cross Linking

Folding of newly synthesized proteins in vivo is believed to be facilitated by the cooperative interaction of a defined group of proteins known as molecular chaperones. We investigated the direct interaction of chaperones with nascent polypeptides in the cytosol of mammalian cells by multiple methods. A new approach using a polyclonal antibody to puromycin allowed us to tag and capture a population of truncated nascent polypeptides with no bias as to the identity of the bound chaperones. In addition, antibodies that recognize the cytosolic chaperones hsp70, CCT (TRiC), hsp40, p48 (Hip), and hsp90 were compared on the basis of their ability to coprecipitate nascent polypeptides, both before and after chemical cross-linking. By all three approaches, hsp70 was found to be the predominant chaperone bound to nascent polypeptides. The interaction between hsp70 and nascent polypeptides is apparently dynamic under physiological conditions but can be stabilized by depletion of ATP or by cross-linking. The cytosolic chaperonin CCT was found to bind primarily to full-length, newly synthesized actin, and tubulin. We demonstrate and caution that nascent polypeptides have a propensity for binding many proteins nonspecifically in cell lysates. Although current models of protein folding in vivo have described additional components in contact with nascent polypeptides, our data indicate that the hsp70 and, perhaps, the hsp90 families are the predominant classes of molecular chaperones that interact with the general population of cytosolic nascent polypeptides.

Retrograde transport of toxins across the endoplasmic reticulum membrane

2003 ◽  
Vol 31 (6) ◽  
pp. 1260-1262 ◽  
Author(s):  
J.M. Lord ◽  
E. Deeks ◽  
C.J. Marsden ◽  
K. Moore ◽  
C. Pateman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Retrograde Transport ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
Endoplasmic Reticulum Membrane ◽  
Cellular Processes ◽  
A Chain ◽  
Toxin Degradation ◽  
Cellular Components

Several protein toxins, including the A chain of the plant protein ricin (RTA), enter mammalian cells by endocytosis and catalytically modify cellular components to disrupt essential cellular processes. In the case of ricin, the process inhibited is protein synthesis. In order to reach their cytosolic substrates, several toxins undergo retrograde transport to the ER (endoplasmic reticulum) before translocating across the ER membrane. To achieve this export, these toxins exploit the ERAD (ER-associated protein degradation) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates in order to intoxicate the cell. Toxins that translocate from the ER have an unusually low lysine content that reduces the likelihood of ubiquitination and ubiquitin-mediated proteasomal degradation. We have changed the two lysyl residues normally present in RTA to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect on the protein, suggesting that the endogenous lysyl residues are not sites for ubiquitin attachment. However, when four additional lysyl residues were introduced into RTA in a way that did not compromise the activity, structure or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasomal inhibitor lactacystin increased the cytotoxicity of the lysine-enriched RTA to a level approaching that of wild-type RTA.

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