scholarly journals Sequence-Independent Targeting of Transmembrane Proteins Synthesized within Vaccinia Virus Factories to Nascent Viral Membranes

2007 ◽  
Vol 81 (6) ◽  
pp. 2646-2655 ◽  
Author(s):  
Matloob Husain ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Structural Features ◽  
Specific Amino Acid ◽  
Viral Membrane ◽  
Virus Factory

ABSTRACT The primary membrane of vaccinia virus, as well as those of other poxviruses, forms within a discrete cytoplasmic factory region. We recently determined the existence of an operative pathway from the endoplasmic reticulum within the virus factory to nascent viral membranes and demonstrated that a viral protein could be diverted from this pathway to Golgi membranes by the addition of COPII-binding sites (M. Husain, A. S. Weisberg, and B. Moss, Proc. Natl. Acad. Sci. USA, 103:19506-19511, 2006). Here we describe an investigation of the structural features that are required for transit of proteins to the viral membrane. Deletion of either the N-terminal domain or the C-terminal cytoplasmic tail from the conserved A9 protein did not prevent its incorporation into viral membranes, whereas deletion of the transmembrane domain resulted in its distribution throughout the cytoplasm. Nevertheless, replacement of the A9 transmembrane domain with the corresponding region of a nonpoxvirus transmembrane protein or of a vaccinia virus extracellular envelope protein allowed viral membrane targeting, indicating no requirement for a specific amino acid sequence. Remarkably, the epitope-tagged A9 transmembrane domain alone, as well as a heterologous transmembrane domain lacking a poxvirus sequence, was sufficient for viral membrane association. The data are consistent with a sequence-independent pathway in which transmembrane proteins that are synthesized within the virus factory and lack COPII or other binding sites that enable conventional endoplasmic reticulum exiting are incorporated into nascent viral membranes.

scholarly journals Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants

2017 ◽  
Vol 114 (51) ◽  
pp. E11001-E11009 ◽  
Author(s):  
Andrea S. Weisberg ◽  
Liliana Maruri-Avidal ◽  
Himani Bisht ◽  
Bryan T. Hansen ◽  
Cindi L. Schwartz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Virus Assembly ◽  
Transmembrane Protein ◽  
Membrane Dynamics ◽  
Deletion Mutants ◽  
Viral Membrane ◽  
Transmission Electron

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

scholarly journals Vaccinia Virus A26 and A27 Proteins Form a Stable Complex Tethered to Mature Virions by Association with the A17 Transmembrane Protein

2008 ◽  
Vol 82 (24) ◽  
pp. 12384-12391 ◽  
Author(s):  
Amanda R. Howard ◽  
Tatiana G. Senkevich ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Matrix Protein ◽  
Transmembrane Domain ◽  
Noncovalent Complex ◽  
Transmembrane Protein ◽  
Amino Acid Identity ◽  
Noncovalent Interaction ◽  
Terminal Segment ◽  
Stable Complex ◽  
Cowpox Virus

ABSTRACT During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.

Evi-2, a common integration site involved in murine myeloid leukemogenesis

1990 ◽  
Vol 10 (9) ◽  
pp. 4658-4666
Author(s):  
A M Buchberg ◽  
H G Bedigian ◽  
N A Jenkins ◽  
N G Copeland
Keyword(s):  
Leucine Zipper ◽  
Transmembrane Domain ◽  
Integration Site ◽  
Transmembrane Protein ◽  
Structural Features ◽  
Viral Integration ◽  
Integration Sites ◽  
Leukemia Viruses ◽  
Common Integration Site

BXH-2 mice have the highest incidence of spontaneous retrovirally induced myeloid leukemia of any known inbred strain and, as such, represent a valuable model system for identifying cellular proto-oncogenes involved in myeloid disease. Chronic murine leukemia viruses often induce disease by insertional activation or mutation of cellular proto-oncogenes. These loci are identified as common viral integration sites in tumor DNAs. Here we report on the characterization of a novel common viral integration site in BXH-2 myeloid leukemias, designated Evi-2. Within the cluster of viral integration sites that define Evi-2, we identified a gene that has the potential for encoding a novel protein of 223 amino acids. This putative proto-oncogene possesses all of the structural features of a transmembrane protein. Within the transmembrane domain is a "leucine zipper," suggesting that Evi-2 is involved in either homopolymer or heteropolymer formation, which may play an important role in the normal functioning of Evi-2. Interestingly, the human homolog of Evi-2 has recently been shown to be tightly linked to the von Recklinghausen neurofibromatosis locus, suggesting a role for Evi-2 in human disease as well.

scholarly journals Cotranslational Intersection between the SRP and GET Targeting Pathways to the Endoplasmic Reticulum of Saccharomyces cerevisiae

2016 ◽  
Vol 36 (18) ◽  
pp. 2374-2383 ◽  
Author(s):  
Ying Zhang ◽  
Thea Schäffer ◽  
Tina Wölfle ◽  
Edith Fitzke ◽  
Gerhard Thiel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Signal Recognition Particle ◽  
Transmembrane Proteins ◽  
Signal Recognition ◽  
Ribosome Binding ◽  
Channel Proteins ◽  
Membrane Spanning ◽  
Combined Data

Targeting of transmembrane proteins to the endoplasmic reticulum (ER) proceeds via either the signal recognition particle (SRP) or the guided entry of tail-anchored proteins (GET) pathway, consisting of Get1 to -5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single C-terminal transmembrane domain termed the tail anchor. Here, we dissect the roles of the SRP and GET pathways in the sorting of homologous, two-membrane-spanning K+channel proteins termed Kcv, Kesv, and Kesv-VV. We show that Kcv is targeted to the ER cotranslationally via its N-terminal transmembrane domain, while Kesv-VV is targeted posttranslationally via its C-terminal transmembrane domain, which recruits Get4-5/Sgt2 and Get3. Unexpectedly, nascent Kcv recruited not only SRP but also the Get4-5 module of the GET pathway to ribosomes. Ribosome binding of Get4-5 was independent of Sgt2 and was strongly outcompeted by SRP. The combined data indicate a previously unrecognized cotranslational interplay between the SRP and GET pathways.

scholarly journals Multiple C2 domain-containing transmembrane proteins promote lipid droplet biogenesis and growth at specialized ER subdomains

2021 ◽  
pp. mbc.E20-09-0590
Author(s):  
Amit S. Joshi ◽  
Joey V. Ragusa ◽  
William A. Prinz ◽  
Sarah Cohen
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Transmembrane Domain ◽  
Neutral Lipids ◽  
Transmembrane Proteins ◽  
C2 Domain ◽  
General Role ◽  
C2 Domains ◽  
Contact Sites ◽  
Er Membrane

Lipid droplets (LDs) are neutral lipid-containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain-containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain (TMD) of these proteins is similar to a reticulon homology domain. Like reticulons, these proteins tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the MCTP TMDs promote LD biogenesis, increasing LD number. MCTPs co-localize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER-LD contact sites. Together, our data indicate that MCTPs form microdomains within ER tubules that regulate LD biogenesis, size, and ER-LD contacts. Interestingly, MCTP punctae colocalized with other organelles as well, suggesting that these proteins may play a more general role in linking tubular ER to organelle contact sites. [Media: see text] [Media: see text]

scholarly journals Sec12p requires Rer1p for sorting to coatomer (COPI)-coated vesicles and retrieval to the ER

1997 ◽  
Vol 110 (8) ◽  
pp. 991-1003 ◽  
Author(s):  
J. Boehm ◽  
F. Letourneur ◽  
W. Ballensiefen ◽  
D. Ossipov ◽  
C. Demolliere ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Retrieval Process ◽  
Hybrid Proteins ◽  
Er Retention ◽  
Golgi Compartment

In Saccharomyces cerevisiae cells lacking the Rer1 protein (Rer1p), the type II transmembrane protein Sec12p fails to be retained in the ER. The transmembrane domain of Sec12p is sufficient to confer Rer1p-dependent ER retention to other membrane proteins. In rer1 mutants a large part of the Sec12-derived proteins can escape to the late Golgi. In contrast, rer3 mutants accumulate Sec12-derived hybrid proteins carrying early Golgi modifications. We found that rer3 mutants harbour unique alleles of the alpha-COP-encoding RET1 gene. ret1 mutants, along with other coatomer mutants, fail to retrieve KKXX-tagged type I transmembrane proteins from the Golgi back to the ER. Surprisingly rer3-11(=ret1-12) mutants do not affect this kind of ER recycling. Pulse-chase experiments using these mutants show that alpha-COP and Rer1p function together in a very early Golgi compartment to initiate the recycling of Sec12p-derived hybrid proteins. Rer1p protein may be directly involved in the retrieval process since it also recycles between the early Golgi and ER in a coatomer (COPI)-dependent manner. Rer1p may act as an adapter coupling the recycling of non-KKXX transmembrane proteins like Sec12p to the coatomer (COPI)-mediated backward traffic.

scholarly journals Bag6 complex contains a minimal tail-anchor–targeting module and a mock BAG domain

2014 ◽  
Vol 112 (1) ◽  
pp. 106-111 ◽  
Author(s):  
Jee-Young Mock ◽  
Justin William Chartron ◽  
Ma’ayan Zaslaver ◽  
Yue Xu ◽  
Yihong Ye ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Endoplasmic Reticulum ◽  
Complex Network ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Degradation Pathway ◽  
Tetratricopeptide Repeat ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Structural Insight ◽  
Insight Into

BCL2-associated athanogene cochaperone 6 (Bag6) plays a central role in cellular homeostasis in a diverse array of processes and is part of the heterotrimeric Bag6 complex, which also includes ubiquitin-like 4A (Ubl4A) and transmembrane domain recognition complex 35 (TRC35). This complex recently has been shown to be important in the TRC pathway, the mislocalized protein degradation pathway, and the endoplasmic reticulum-associated degradation pathway. Here we define the architecture of the Bag6 complex, demonstrating that both TRC35 and Ubl4A have distinct C-terminal binding sites on Bag6 defining a minimal Bag6 complex. A crystal structure of the Bag6–Ubl4A dimer demonstrates that Bag6–BAG is not a canonical BAG domain, and this finding is substantiated biochemically. Remarkably, the minimal Bag6 complex defined here facilitates tail-anchored substrate transfer from small glutamine-rich tetratricopeptide repeat-containing protein α to TRC40. These findings provide structural insight into the complex network of proteins coordinated by Bag6.

scholarly journals A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1

2004 ◽  
Vol 167 (3) ◽  
pp. 445-456 ◽  
Author(s):  
Yukio Kimata ◽  
Daisuke Oikawa ◽  
Yusuke Shimizu ◽  
Yuki Ishiwata-Kimata ◽  
Kenji Kohno
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Binding Site ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Sensory System ◽  
Type I ◽  
Stress Signal ◽  
Stress Sensing ◽  
The Unfolded Protein Response

In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses.

scholarly journals Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

2001 ◽  
Vol 12 (7) ◽  
pp. 2031-2046 ◽  
Author(s):  
Nina Tolonen ◽  
Laura Doglio ◽  
Sibylle Schleich ◽  
Jacomine Krijnse Locker
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Nuclear Envelope Assembly ◽  
Green Fluorescent Protein Tagging ◽  
Green Fluorescent

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ∼45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [3H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

scholarly journals FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Fang Chen ◽  
Bing Yan ◽  
Jie Ren ◽  
Rui Lyu ◽  
Yanfang Wu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Energy Metabolism ◽  
Molecular Basis ◽  
Lipid Droplets ◽  
Adipocyte Differentiation ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Cytoskeletal Protein ◽  
Interacting Proteins

Lipid droplets (LDs) are critical for lipid storage and energy metabolism. LDs form in the endoplasmic reticulum (ER). However, the molecular basis for LD biogenesis remains elusive. Here, we show that fat storage–inducing transmembrane protein 2 (FIT2) interacts with ER tubule-forming proteins Rtn4 and REEP5. The association is mainly transmembrane domain based and stimulated by oleic acid. Depletion of ER tubule-forming proteins decreases the number and size of LDs in cells and Caenorhabditis elegans, mimicking loss of FIT2. Through cytosolic loops, FIT2 binds to cytoskeletal protein septin 7, an interaction that is also required for normal LD biogenesis. Depletion of ER tubule-forming proteins or septins delays nascent LD formation. In addition, FIT2-interacting proteins are up-regulated during adipocyte differentiation, and ER tubule-forming proteins, septin 7, and FIT2 are transiently enriched at LD formation sites. Thus, FIT2-mediated nascent LD biogenesis is facilitated by ER tubule-forming proteins and septins.

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