scholarly journals Glycosylation Can Influence Topogenesis of Membrane Proteins and Reveals Dynamic Reorientation of Nascent Polypeptides within the Translocon

1999 ◽  
Vol 147 (2) ◽  
pp. 257-266 ◽  
Author(s):  
Veit Goder ◽  
Christoph Bieri ◽  
Martin Spiess
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Dynamic Process ◽  
Membrane Targeting ◽  
Protein Orientation ◽  
Nascent Polypeptide ◽  
Targeting Efficiency ◽  
Internal Signal ◽  
Chimeric Model

The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH2-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH2-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.

scholarly journals Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

2016 ◽  
Vol 113 (7) ◽  
pp. E829-E838 ◽  
Author(s):  
Yuhei Chadani ◽  
Tatsuya Niwa ◽  
Shinobu Chiba ◽  
Hideki Taguchi ◽  
Koreaki Ito
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Ribosome Profiling ◽  
Cytosolic Proteins ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Chemical Trait

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study,Escherichia coliSecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members ofE.colithat were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl–tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.

scholarly journals Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene.

1992 ◽  
Vol 3 (2) ◽  
pp. 143-155 ◽  
Author(s):  
D T Ng ◽  
S S Watowich ◽  
R A Lamb
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Complex Formation ◽  
Simian Virus ◽  
Single Site ◽  
Simian Virus 5 ◽  
Chaperone Protein ◽  
Protein Molecules ◽  
Mutant Polypeptides

The endoplasmic reticulum (ER)-localized chaperone protein, GRP78-BiP, is involved in the folding and oligomerization of secreted and membrane proteins, including the simian virus 5 hemagglutinin-neuraminidase (HN) glycoprotein. To understand this interaction better, we have constructed a series of HN mutants in which specific portions of the extracytoplasmic domain have been deleted. Analysis of these mutant polypeptides expressed in CV-1 cells have indicated that GRP78-BiP binds to selective sequences in HN and that there exists more than a single site of interaction. Mutant polypeptides have been characterized that are competent and incompetent for association with GRP78-BiP. These mutants have been used to show that the induction of GRP78-BiP synthesis due to the presence of nonnative protein molecules in the ER is dependent on GRP78-BiP complex formation with its substrates. These studies have implications for the function of the GRP78-BiP protein and the mechanism by which the gene is regulated.

scholarly journals A Cdc48 “Retrochaperone” Function Is Required for the Solubility of Retrotranslocated, Integral Membrane Endoplasmic Reticulum-associated Degradation (ERAD-M) Substrates

2017 ◽  
Vol 292 (8) ◽  
pp. 3112-3128 ◽  
Author(s):  
Sonya Neal ◽  
Raymond Mak ◽  
Eric J. Bennett ◽  
Randolph Hampton
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Full Length ◽  
26S Proteasome ◽  
Polyubiquitin Chains ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Erad Pathway ◽  
Er Membrane

A surprising feature of endoplasmic reticulum (ER)-associated degradation (ERAD) is the movement, or retrotranslocation, of ubiquitinated substrates from the ER lumen or membrane to the cytosol where they are degraded by the 26S proteasome. Multispanning ER membrane proteins, called ERAD-M substrates, are retrotranslocated to the cytosol as full-length intermediates during ERAD, and we have investigated how they maintain substrate solubility. Using an in vivo assay, we show that retrotranslocated ERAD-M substrates are moved to the cytoplasm as part of the normal ERAD pathway, where they are part of a solely proteinaceous complex. Using proteomics and direct biochemical confirmation, we found that Cdc48 serves as a critical “retrochaperone” for these ERAD-M substrates. Cdc48 binding to retrotranslocated, ubiquitinated ERAD-M substrates is required for their solubility; removal of the polyubiquitin chains or competition for binding by addition of free polyubiquitin liberated Cdc48 from retrotranslocated proteins and rendered them insoluble. All components of the canonical Cdc48 complex Cdc48-Npl4-Ufd1 were present in solubilized ERAD-M substrates. This function of the complex was observed for both HRD and DOA pathway substrates. Thus, in addition to the long known ATP-dependent extraction of ERAD substrates during retrotranslocation, the Cdc48 complex is generally and critically needed for the solubility of retrotranslocated ERAD-M intermediates.

scholarly journals A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon

2016 ◽  
Vol 113 (39) ◽  
pp. 10902-10907 ◽  
Author(s):  
Emily Breeze ◽  
Natasha Dzimitrowicz ◽  
Verena Kriechbaumer ◽  
Rhiannon Brooks ◽  
Stanley W. Botchway ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Structural Feature ◽  
Mechanism Of Action ◽  
Membrane Curvature ◽  
Amphipathic Helix ◽  
Transmembrane Topology ◽  
C Terminus ◽  
Er Membrane

Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.

scholarly journals Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

1998 ◽  
Vol 9 (1) ◽  
pp. 103-115 ◽  
Author(s):  
Andrea Neuhof ◽  
Melissa M. Rolls ◽  
Berit Jungnickel ◽  
Kai-Uwe Kalies ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Membrane Binding ◽  
Membrane Targeting ◽  
Signal Recognition ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Cytosolic Factors ◽  
Er Membrane

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

scholarly journals Stress-Associated Endoplasmic Reticulum Protein 1 (Serp1)/Ribosome-Associated Membrane Protein 4 (Ramp4) Stabilizes Membrane Proteins during Stress and Facilitates Subsequent Glycosylation

1999 ◽  
Vol 147 (6) ◽  
pp. 1195-1204 ◽  
Author(s):  
Atsushi Yamaguchi ◽  
Osamu Hori ◽  
David M. Stern ◽  
Enno Hartmann ◽  
Satoshi Ogawa ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Er Stress ◽  
Artery Occlusion ◽  
Integral Membrane Proteins ◽  
Endoplasmic Reticulum Protein ◽  
Cerebral Artery Occlusion

Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66–amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61α and Sec61β, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61α and Sec61β, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

scholarly journals Active translocon complexes labeled with GFP–Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum

2002 ◽  
Vol 158 (3) ◽  
pp. 497-506 ◽  
Author(s):  
Andrei V. Nikonov ◽  
Erik Snapp ◽  
Jennifer Lippincott-Schwartz ◽  
Gert Kreibich
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Membrane Proteins ◽  
Effective Diffusion ◽  
Diffusion Constant ◽  
Temperature Sensitive ◽  
Lateral Mobility ◽  
Nascent Polypeptide ◽  
Membrane Bound ◽  
Polypeptide Chains

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP–Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP–Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP–Dad1 in the ER membranes, but to a level that is still lower than that of free GFP–Dad1. This suggests that GFP–Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.

scholarly journals The Efficiency of Protein Compartmentalization into the Secretory Pathway

2005 ◽  
Vol 16 (1) ◽  
pp. 279-291 ◽  
Author(s):  
Corinna G. Levine ◽  
Devarati Mitra ◽  
Ajay Sharma ◽  
Carolyn L. Smith ◽  
Ramanujan S. Hegde
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Cellular Factors ◽  
Specific Manner ◽  
In Trans ◽  
Mature Domain ◽  
In Cis

Numerous proteins targeted for the secretory pathway are increasingly implicated in functional or pathological roles at alternative cellular destinations. The parameters that allow secretory or membrane proteins to reside in intracellular locales outside the secretory pathway remain largely unexplored. In this study, we have used an extremely sensitive and quantitative assay to measure the in vivo efficiency of signal sequence-mediated protein segregation into the secretory pathway. Our findings reveal that segregation efficiency varies tremendously among signals, ranging from >95 to <60%. The nonsegregated fraction is generated by a combination of mechanisms that includes inefficient signal-mediated translocation into the endoplasmic reticulum and leaky ribosomal scanning. The segregation efficiency of some, but not other signal sequences, could be influenced in cis by residues in the mature domain or in trans by yet unidentified cellular factors. These findings imply that protein compartmentalization can be modulated in a substrate-specific manner to generate biologically significant quantities of cytosolically available secretory and membrane proteins.

An Ultra-Structural Study of Human Melanoma in Vivo and Vitro

1976 ◽  
Vol 34 ◽  
pp. 258-259
Author(s):  
James R. Gaylor ◽  
Fredda Schafer ◽  
Robert E. Nordquist
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Protein Aggregation ◽  
Structural Study ◽  
Human Melanoma ◽  
Protein Matrix ◽  
Early Form ◽  
Endoplasmic Reticulum Protein ◽  
Mitochondrial Origin

Several theories on the origin of the melanosome exist. These include the Golgi origin theory, in which a tyrosinase-rich protein is "packaged" by the Golgi apparatus, thus forming the early form of the melanosome. A second theory postulates a mitochondrial origin of melanosomes. Its author contends that the melanosome is a modified mitochondria which acquires melanin during its development. A third theory states that a pre-melanosome is formed in the smooth or rough endoplasmic reticulum. Protein aggregation is suggested by one author as a possible source of the melanosome. This fourth theory postulates that the melanosome originates when the protein products of several genetic loci aggregate in the cytoplasm of the melanocyte. It is this protein matrix on which the melanin is deposited. It was with these theories in mind that this project was undertaken.

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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