Membrane-integration Characteristics of Two ABC Transporters, CFTR and P-glycoprotein

Enquist, Karl; Fransson, Mawritz; Boekel, Carolina; Bengtsson, Inger; Geiger, Karin; Lang, Lisa; Pettersson, Aron; Johansson, Sofia; Heijne, Gunnar von; Nilsson, IngMarie

doi:10.1016/j.jmb.2009.02.035

Cited by 50 publications

(54 citation statements)

References 70 publications

(95 reference statements)

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“…Thus, we speculated that some type of helix-helix interaction stabilizes the insertion of HR2 to keep an N cyt /C cyt 2B topology. It has been shown recently that in some cases the insertion of poorly hydrophobic regions depends on the presence of neighboring loops and/or TM segments (20), especially in the case of the preceding TM segment (23,55). In our attempts to unravel the disposition of 2B in biological membranes, we focused on the insertion of truncated C-terminal reporter tag fusions.…”

Section: Resultsmentioning

confidence: 99%

Membrane Integration of Poliovirus 2B Viroporin

Martínez-Gil

Bañó‐Polo

Redondo

et al. 2011

J Virol

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Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of in vitro translation-glycosylation compatible with the transloconmediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the in vitro translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes.Virus infections can lead to a variety of cellular injuries, and usually these involve the restructuring of host membrane systems. Viroporins are a group of small virally encoded proteins that interact with cellular membranes to modify permeability and promote the release of viral particles. A typical feature exhibited by viroporins is the presence of at least one membrane-spanning helix anchoring the protein into membranes. After membrane insertion, their oligomerization creates hydrophilic channels or pores (22).Poliovirus is the enterovirus prototype member of the Picornaviridae family. This small, nonenveloped, icosahedral virus possesses a single-stranded 7.5-kb positive-sense RNA genome that encodes a single polyprotein. Polyprotein processing by virus-encoded proteases yields the structural P1 region proteins that encapsidate viral RNA and the nonstructural P2 and P3 region proteins involved in the replication of the viral RNA and membrane permeabilization (2). Nonstructural 2B protein is one of the products generated on processing the P2 region (62). Viroporin 2B has been identified as one of the viral proteins responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. Different 2B proteins expressed in cells have been localized at the endoplasmic reticulum (ER), Golgi complex, and, to a lesser extent, to the plasma and mitochondrial membranes (18,31,49,58). Biochemical and structural data indicate that viroporins form homo-oligomers that create pores in the ER and Golgi complex membranes (1,16,17,30,59). However, experimental data dealing with the mechanism of the membrane integration of the 2B product are lacking to date.The poliovirus 2B viroporin protein is hydrophobic overall and rather small (97 amino acids). Hydrophobicity within the viroporin 2B sequence seems to cluster in two main regions (F...

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Section: Resultsmentioning

confidence: 99%

Membrane Integration of Poliovirus 2B Viroporin

Martínez-Gil

Bañó‐Polo

Redondo

et al. 2011

J Virol

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“…It has been shown previously that in some cases, a neighboring TMH can favor the membrane insertion of a mTMH 17 and that there is a correlation between the polarity of a TMH and its interaction area with the rest of the protein. 12 The comprehensive collection of mTMHs studied here is, on average, found to be more buried than other TMHs (p b 2 × 10 − 6 as judged by a two-sided t test; data not shown).…”

Section: Membrane Insertion Of Mtmhs In the Presence Of Flanking Tranmentioning

confidence: 96%

Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context

Hedin

Öjemalm

Bernsel

et al. 2010

Journal of Molecular Biology

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119

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In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.

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“…The K app value can be converted into the apparent free energy difference between the non-inserted state and the inserted state using: G app = -RTlnK app , where R is the gas constant (R = 1.986 kcal/K·mol) and T is the absolute temperature (T = 303 K). It has been shown previously that in some cases, a neighboring TMH can favor the membrane insertion of poorly hydrophobic TM segments 23 , especially in the case of the preceding TM segment 24 .…”

Section: Membrane Insertion Of Isolated Tm Segmentsmentioning

confidence: 96%

Membrane Insertion and Topology of the Translocating Chain-Associating Membrane Protein (TRAM)

Tamborero

Vilar

Martínez-Gil

et al. 2011

Journal of Molecular Biology

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The translocating chain-associating membrane protein (TRAM) is a glycoprotein involved in the translocation of secreted proteins into the ER lumen, and in the insertion of integral membrane proteins into the lipid bilayer. As a major step toward elucidating the structure of the functional endoplasmic reticulum (ER) translocation/insertion machinery, we have characterized the membrane integration mechanism and the transmembrane (TM) topology of TRAM using two approaches: photocross-linking and truncated C-terminal reporter tag fusions. Our data indicate that TRAM is recognized by the signal recognition particle (SRP) and translocon components, and suggest a membrane topology with eight TM segments, including several poorly hydrophobic segments. Furthermore, we studied the membrane insertion capacity of these poorly hydrophobic segments into the ER membrane by themselves. Finally, we confirmed the main features of the proposed membrane topology in mammalian cells expressing full-length TRAM. MEMBRANE INSERTION AND TOPOLOGY OF THE TRANSLOCATING CHAIN-ASSOCIATING MEMBRANE PROTEIN (TRAM)Silvia AbstractThe translocating chain-associating membrane protein (TRAM) is a glycoprotein involved in the translocation of secreted proteins into the ER lumen, and in the insertion of integral membrane proteins into the lipid bilayer. As a major step toward elucidating the structure of the functional endoplasmic reticulum (ER) translocation/insertion machinery, we have characterized the membrane integration mechanism and the transmembrane (TM) topology of TRAM using two approaches: photocross-linking and truncated C-terminal reporter tag fusions. Our data indicate that TRAM is recognized by the signal recognition particle (SRP) and translocon components, and suggest a membrane topology with eight TM segments, including several poorly hydrophobic segments. Furthermore, we studied the membrane insertion capacity of these poorly hydrophobic segments into the ER membrane by themselves. Finally, we confirmed the main features of the proposed membrane topology in mammalian cells expressing full-length TRAM.

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Membrane-integration Characteristics of Two ABC Transporters, CFTR and P-glycoprotein

Cited by 50 publications

References 70 publications

Membrane Integration of Poliovirus 2B Viroporin

Membrane Integration of Poliovirus 2B Viroporin

Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context

Membrane Insertion and Topology of the Translocating Chain-Associating Membrane Protein (TRAM)

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