Role of Ribosomes in Reinitiation of Membrane Insertion of Internal Transmembrane Segments in a Polytopic Membrane Protein

Wang, Changsen; Chen, Mingang; Han, Ernest; Zhang, Jian-Ting

doi:10.1021/bi970041g

“…This observation suggests that there may be a ribosome re-targeting process involved in TM2 membrane translocation following TM1 membrane translocation. A similar observation has been made about the role of ribosomes in the membrane translocation of Pgp sequences [39,50,51]. It is therefore possible that following the membrane translocation of TM1, ribosomes release from the protein-conducting channel and redock back when TM2 emerges from the ribosome.…”

Section: Discussionsupporting

confidence: 59%

Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein

Zhang

¹

2000

Biochemical Journal

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Multidrug-resistance-associated protein (MRP) is a member of the ATP-binding cassette (ABC) membrane-transport superfamily and is responsible for multidrug resistance in cancer cells. Distinct from other members of the ABC superfamily, MRP has three membrane-spanning domains (MSDs) and the N-terminus is located extracellularly. It has been shown that the first MSD (MSD1) with an extracellular N-terminus is important for MRP function. To address what ensures the generation of this structural organization of MRP and to understand in general the molecular mechanism of membrane folding of polytopic proteins with extracellular N-termini, the biogenesis of MSD1 in human MRP1 was examined using an in vitro expression system. Surprisingly, the second transmembrane segment (TM2) in MSD1 was found to play a critical role in the correct membrane translocation and folding of MSD1 in human MRP1. TM2 not only plays an essential role to ensure the N-terminus-outside/C-terminus-inside orientation of TM1 with an extracellular N-terminus, it can also translocate into membranes post-translationally in a signal-recognition particle and ribosome-dependent manner to provide an additional insurance for correct folding of MSD1 in MRP. These findings suggest that TM2 in a polytopic membrane protein with an extracellular N-terminus may play a critical role in controlling correct membrane translocation and folding of the protein in general.

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“…Translation using pre-mixed RRL and WGE were performed as described previously [38]. Translation in RRL in the presence of wheat germ ribosome (WGR) and purification of WGR were done in the same way as described by Wang et al [39]. The fluorograph images were digitized using a HP ScanJet 6100C and Adobe Photoshop 4.0.…”

Section: Transcription and Translation In Vitromentioning

confidence: 99%

“…In previous studies, it has been reported that ribosomes are important for the membrane targeting and translocation of polytopic membrane proteins [39]. Because TM2 of human MRP1 is a very important element in the topogenesis of MSD1, MRP-N2R was translated in both RRL or WGE to determine whether the membrane translocation and orientation of MRP-N2R is different between these two different systems.…”

Section: Ribosomal Role In Membrane Translocation Of Msd1mentioning

confidence: 99%

Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein

ZHANG¹

2000

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Multidrug-resistance-associated protein (MRP) is a member of the ATP-binding cassette (ABC) membrane-transport superfamily and is responsible for multidrug resistance in cancer cells. Distinct from other members of the ABC superfamily, MRP has three membrane-spanning domains (MSDs) and the N-terminus is located extracellularly. It has been shown that the first MSD (MSD1) with an extracellular N-terminus is important for MRP function. To address what ensures the generation of this structural organization of MRP and to understand in general the molecular mechanism of membrane folding of polytopic proteins with extracellular N-termini, the biogenesis of MSD1 in human MRP1 was examined using an in vitro expression system. Surprisingly, the second transmembrane segment (TM2) in MSD1 was found to play a critical role in the correct membrane translocation and folding of MSD1 in human MRP1. TM2 not only plays an essential role to ensure the N-terminus-outside/C-terminus-inside orientation of TM1 with an extracellular N-terminus, it can also translocate into membranes post-translationally in a signal-recognition particle and ribosome-dependent manner to provide an additional insurance for correct folding of MSD1 in MRP. These findings suggest that TM2 in a polytopic membrane protein with an extracellular N-terminus may play a critical role in controlling correct membrane translocation and folding of the protein in general.

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“…For the analysis of C-half topogenesis in the presence of wheat-germ ribosomes, we isolated wheat-germ ribosomes from crude wheat-germ extracts by centrifugation as described by Wang et al (23). Approximately 6 µg of ribosomes was added to the rabbit reticulocyte lysate translation mix to a final concentration of 10% (v/v).…”

Section: Methodsmentioning

confidence: 99%

“…Wheat-Germ Ribosomes Alter the Generation of Two Topological Orientations for the C-Half of Pgp. Recently, we have found that ribosomes are involved in the membrane insertion process for the N-half sequence of Pgp (23). To test whether ribosomes also affect the topogenesis of C-half Pgp, we tested the effect of wheat-germ ribosomes on the topological orientation of various Pgp constructs.…”

Section: T H I S C O N T E N T Imentioning

confidence: 99%

Mechanism Involved in Generating the Carboxyl-Terminal Half Topology of P-Glycoprotein

Han

¹

,

Zhang

²

1998

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P-Glycoprotein (Pgp) is a polytopic membrane protein that consists of a tandem repeat of a transmembrane (TM) domain followed by a nucleotide-binding domain. For the carboxyl-terminal half (C-half) of Pgp, at least three different topological orientations have been observed. One major difference between these topologies is reflected in the membrane insertion property of TM8, which is predicted to (1) function as a stop-transfer sequence, (2) lack stop-transfer activity, or (3) function as a signal-anchor sequence. To understand the mechanism involved in generating multiple topological forms for the C-half of Pgp, we investigated the membrane insertion properties of TM segments using the Chinese hamster pgp1 Pgp as a model protein in a cell-free system. We found that TM8 alone or in the presence of TM7 functions as a signal-anchor sequence to insert into membranes with a cytoplasmic amino terminus and an extra-cytoplasmic carboxyl terminus. However, TM8 displayed stop-transfer activity when linked to the C-terminal end of the signal-anchor sequence, TM1. In addition, the membrane orientation of TM8 was found to be regulated by the charge distribution flanking TM8. Interestingly, we found that mammalian and wheat germ ribosomes differentially regulate the signal-anchor and stop-transfer properties of TM8. We conclude that the unique topogenic properties of TM8 direct the generation of multiple C-half topological orientations.

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Role of Ribosomes in Reinitiation of Membrane Insertion of Internal Transmembrane Segments in a Polytopic Membrane Protein

Cited by 5 publications

References 32 publications

Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein

Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein

Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein

Mechanism Involved in Generating the Carboxyl-Terminal Half Topology of P-Glycoprotein

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