scholarly journals Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein

2002 ◽  
Vol 13 (12) ◽  
pp. 4114-4129 ◽  
Author(s):  
Suzanna L. Meacock ◽  
Fabienne J.L. Lecomte ◽  
Samuel G. Crawshaw ◽  
Stephen High
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Polypeptide Chain ◽  
Transmembrane Domain ◽  
Insertion Site ◽  
Transmembrane Domains ◽  
Membrane Insertion ◽  
Relative Location ◽  
Transmembrane Topology ◽  
Nascent Chain

We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the α and β subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61α and Sec61β during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require theN-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the “stage” of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.

Specific transmembrane segments are selectively delayed at the ER translocon during opsin biogenesis

2008 ◽  
Vol 411 (3) ◽  
pp. 495-506 ◽  
Author(s):  
Nurzian Ismail ◽  
Samuel G. Crawshaw ◽  
Benedict C. S. Cross ◽  
Anna C. Haagsma ◽  
Stephen High
Keyword(s):  
Endoplasmic Reticulum ◽  
Polypeptide Chain ◽  
Cross Linking ◽  
Relative Location ◽  
Intrinsic Properties ◽  
Site Specific ◽  
Transmembrane Segments ◽  
Nascent Chain ◽  
Polytopic Membrane Protein ◽  
A Site

A site-specific cross-linking approach was used to study the integration of TM (transmembrane) segments 4–7 of the polytopic membrane protein, opsin, at the ER (endoplasmic reticulum). We found that although TM4 exits the ER translocon rapidly, TM segments 5, 6 and 7 are all retained at the translocon until opsin biosynthesis is terminated. Furthermore, although artificial extension of the nascent chain is not sufficient to release the C-terminal region of opsin from the translocon, substitution of the native TM segment 7 with a more hydrophobic TM segment results in its rapid lateral exit into the lipid bilayer. We conclude that the intrinsic properties of a TM segment determine the timing of its membrane integration rather than its relative location within the polypeptide chain. A pronounced and prolonged association of opsin TM5 with the translocon-associated component PAT-10 was also observed, suggesting that PAT-10 may facilitate the assembly of distinct opsin subdomains during membrane integration. The results of the present study strongly support a model in which the ER translocon co-ordinates the integration of selected TM segments in response to the specific requirements of the precursor being synthesized.

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.

Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5

2001 ◽  
Vol 281 (1) ◽  
pp. C215-C223 ◽  
Author(s):  
Robert T. Watson ◽  
Jeffrey E. Pessin
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Glucose Transporter ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Snare Protein ◽  
Glut 4 ◽  
Golgi Localization ◽  
Syntaxin 3

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.

scholarly journals Polar Transmembrane Domains Target Proteins to the Interior of the Yeast Vacuole

2000 ◽  
Vol 11 (11) ◽  
pp. 3737-3749 ◽  
Author(s):  
Fulvio Reggiori ◽  
Michael W. Black ◽  
Hugh R. B. Pelham
Keyword(s):  
Endoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Control Mechanisms ◽  
Multiple Points ◽  
Yeast Vacuole ◽  
Late Endosomes ◽  
Vacuolar Protein ◽  
Dependent Pathway ◽  
Endocytic Pathways

Membrane proteins transported to the yeast vacuole can have two fates. Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded. The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3–dependent pathway, which bypasses late endosomes, but the endosomal SNARE Pep12p must avoid it more directly. Deletion analysis revealed no cytoplasmic sequences necessary to prevent the internalization of Pep12p in endosomes. However, introduction of acidic residues into the cytoplasmic half of the transmembrane domain created a dominant internalization signal. In other contexts, this same feature diverted proteins from the Golgi to endosomes and slowed their exit from the endoplasmic reticulum. The more modestly polar transmembrane domains of Sec12p and Ufe1p, which normally serve to hold these proteins in the endoplasmic reticulum, also cause Pep12p to be internalized, as does that of the vacuolar protein Cps1p. It seems that quality control mechanisms recognize polar transmembrane domains at multiple points in the secretory and endocytic pathways and in endosomes sort proteins for subsequent destruction in the vacuole. These mechanisms may minimize the damaging effects of abnormally exposed polar residues while being exploited for the localization of some normal proteins.

scholarly journals Targeting of LRRC59 to the Endoplasmic Reticulum and the Inner Nuclear Membrane

2019 ◽  
Vol 20 (2) ◽  
pp. 334 ◽  
Author(s):  
Marina Blenski ◽  
Ralph Kehlenbach
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Nuclear Import ◽  
Transmembrane Domain ◽  
Membrane Insertion ◽  
Leucine Rich Repeat ◽  
Inner Nuclear Membrane ◽  
Importin Α ◽  
Major Route

LRRC59 (leucine-rich repeat-containing protein 59) is a tail-anchored protein with a single transmembrane domain close to its C-terminal end that localizes to the endoplasmic reticulum (ER) and the nuclear envelope. Here, we investigate the mechanisms of membrane integration of LRRC59 and its targeting to the inner nuclear membrane (INM). Using purified microsomes, we show that LRRC59 can be post-translationally inserted into ER-derived membranes. The TRC-pathway, a major route for post-translational membrane insertion, is not required for LRRC59. Like emerin, another tail-anchored protein, LRRC59 reaches the INM, as demonstrated by rapamycin-dependent dimerization assays. Using different approaches to inhibit importin α/β-dependent nuclear import of soluble proteins, we show that the classic nuclear transport machinery does not play a major role in INM-targeting of LRRC59. Instead, the size of the cytoplasmic domain of LRRC59 is an important feature, suggesting that targeting is governed by passive diffusion.

scholarly journals A cryptic matrix targeting signal of the yeast ADP/ATP carrier normally inserted by the TIM22 complex is recognized by the TIM23 machinery

2004 ◽  
Vol 385 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Maïlys A. S. VERGNOLLE ◽  
Helen SAWNEY ◽  
Tina JUNNE ◽  
Luisita DOLFINI ◽  
Kostas TOKATLIDIS
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Mitochondrial Protein ◽  
Amino Acid Sequences ◽  
Membrane Insertion ◽  
Mitochondrial Targeting ◽  
Terminal Part ◽  
Inner Membrane ◽  
The Matrix ◽  
Targeting Signal

The yeast ADP/ATP carrier (AAC) is a mitochondrial protein that is targeted to the inner membrane via the TIM10 and TIM22 translocase complexes. AAC is devoid of a typical mitochondrial targeting signal and its targeting and insertion are thought to be guided by internal amino acid sequences. Here we show that AAC contains a cryptic matrix targeting signal that can target up to two thirds of the N-terminal part of the protein to the matrix. This event is coordinated by the TIM23 translocase and displays all the features of the matrix-targeting pathway. However, in the context of the whole protein, this signal is ‘masked’ and rendered non-functional as the polypeptide is targeted to the inner membrane via the TIM10 and TIM22 translocases. Our data suggest that after crossing the outer membrane the whole polypeptide chain of AAC is necessary to commit the precursor to the TIM22-mediated inner membrane insertion pathway.

scholarly journals Formation of bovine viral diarrhea virus E1–E2 heterodimers is essential for virus entry and depends on charged residues in the transmembrane domains

2008 ◽  
Vol 89 (9) ◽  
pp. 2114-2121 ◽  
Author(s):  
Saskia Ronecker ◽  
Gert Zimmer ◽  
Georg Herrler ◽  
Irene Greiser-Wilke ◽  
Beatrice Grummer
Keyword(s):  
Amino Acid ◽  
Bovine Viral Diarrhea Virus ◽  
Transmembrane Domain ◽  
Virus Entry ◽  
Transmembrane Domains ◽  
Site Directed Mutagenesis ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

The envelope of bovine viral diarrhea virus (BVDV) contains the glycoproteins Erns, E1 and E2. Complementation of a recombinant vesicular stomatitis virus (VSV) with BVDV glycoproteins resulted in infectious pseudotyped viruses. To elucidate the specific role of each of the single envelope glycoproteins during viral entry, pseudotypes were generated bearing the BVDV envelope proteins in different combinations. Pseudoviruses that contained E1 and E2 but not Erns were infectious, indicating that Erns is dispensable for virus entry. VSV/BVDV pseudotypes with chimeric proteins (the ectodomain of the BVDV glycoprotein and the transmembrane domain of the VSV-G protein) were not infectious. The fact that E1–E2 heterodimers were not detected if one of the proteins was chimeric indicated that the heterodimers are crucial for BVDV entry. It was shown by site-directed mutagenesis that the charged amino acids in the transmembrane domains of BVDV E1 (lysine and arginine) and the charged amino acid in the transmembrane domain of E2 (arginine) play a key role in heterodimer formation. Pseudoviruses bearing the mutation E2-R/A, where the charged amino acid was substituted by alanine, were not infectious, supporting the hypothesis that E1–E2 heterodimers are essential for BVDV entry.

scholarly journals Positive Charges of Translocating Polypeptide Chain Retrieve an Upstream Marginal Hydrophobic Segment from the Endoplasmic Reticulum Lumen to the Translocon

2010 ◽  
Vol 21 (12) ◽  
pp. 2045-2056 ◽  
Author(s):  
Hidenobu Fujita ◽  
Yuichiro Kida ◽  
Masatoshi Hagiwara ◽  
Fumiko Morimoto ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Polypeptide Chain ◽  
Water Environment ◽  
Amino Acid Residues ◽  
Endoplasmic Reticulum Lumen ◽  
Membrane Spanning ◽  
Positively Charged

Positively charged amino acid residues are well recognized topology determinants of membrane proteins. They contribute to the stop-translocation of a polypeptide translocating through the translocon and to determine the orientation of signal sequences penetrating the membrane. Here we analyzed the function of these positively charged residues during stop-translocation in vitro. Surprisingly, the positive charges facilitated membrane spanning of a marginally hydrophobic segment, even when separated from the hydrophobic segment by 70 residues. In this case, the hydrophobic segment was exposed to the lumen, and then the downstream positive charges triggered the segment to slide back into the membrane. The marginally hydrophobic segment spanned the membrane, but maintained access to the water environment. The positive charges not only fix the hydrophobic segment in the membrane at its flanking position, but also have a much more dynamic action than previously realized.

scholarly journals Integration of membrane proteins into the endoplasmic reticulum requires GTP.

1988 ◽  
Vol 107 (1) ◽  
pp. 69-77 ◽  
Author(s):  
C Wilson ◽  
T Connolly ◽  
T Morrison ◽  
R Gilmore
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
G Protein ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Membrane Insertion ◽  
Signal Recognition ◽  
Free System ◽  
Amino Terminal ◽  
Nascent Chain

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.

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.

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