Insertion of the Polytopic Membrane Protein MalF Is Dependent on the Bacterial Secretion Machinery

Traxler, Beth; Murphy, Christian

doi:10.1074/jbc.271.21.12394

Cited by 97 publications

(106 citation statements)

References 26 publications

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“…It was first suggested that MalF inserted into the cytoplasmic membrane in a Sec-independent fashion (McGovern and Beckwith, 1991). Recently, two reports have shown that a strong SecE depletion prevented the correct insertion of MalF (Jander et al, 1996;Traxler and Murphy, 1996). Interestingly, these authors favour the idea that it is the L3 loop that might use the Sec apparatus for translocation into the periplasm.…”

Section: Discussionmentioning

confidence: 99%

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli

Mourez

Skouloubris

Betton

et al. 1997

Molecular Microbiology

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SummaryWe analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli. We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, 24 , involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.

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Section: Discussionmentioning

confidence: 99%

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli

Mourez

Skouloubris

Betton

et al. 1997

Molecular Microbiology

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“…The role of SecA in the complete assembly of inner membrane proteins is difficult to study in vivo and may differ for different substrate proteins (25)(26)(27). It should be noted that full-length FtsQ did not integrate into SecYEG proteoliposomes either in the presence or absence of SecA (data not shown) suggesting that additional components are required for complete in vitro reconstitution of inner membrane protein biogenesis.…”

Section: Seca Is Not Required For the Srp-dependent Targeting And Memmentioning

confidence: 97%

SecA Is Not Required for Signal Recognition Particle-mediated Targeting and Initial Membrane Insertion of a Nascent Inner Membrane Protein

Scotti¹,

Valent²,

Manting

et al. 1999

Journal of Biological Chemistry

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In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosomenascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.Going across or integrating into the inner membrane presents two different challenges to proteins synthesized in the cytosol of a prokaryotic cell, and this is reflected by the existence of two main targeting routes. The SecB pathway is specialized for the targeting of periplasmic and outer membrane proteins. SecB is a cytosolic chaperone that binds to the mature region of a subset of preproteins (1). The SecB-preprotein complex is targeted to SecA, which is bound with high affinity to the membrane-embedded SecYEG complex (for review, see Ref.2).Both in vivo and in vitro experiments indicate that the signal recognition particle (SRP) 1 pathway is primarily used for the targeting of integral inner membrane proteins (3-7). This pathway resembles SRP-mediated targeting of proteins to the membrane of the endoplasmic reticulum (ER) in eukaryotes (for review, see Ref. 8). The eukaryotic SRP interacts with nascent membrane and secreted proteins and targets them to the SRP receptor SR␣ at the ER membrane. The eukaryotic SRP is a complex consisting of six proteins arranged on an RNA scaffold, the 7 S RNA. Escherichia coli contains a smaller SRP composed of the P48 protein and the 4.5 S RNA, which are homologous to the eukaryotic SRP54 and the 7 S RNA, respectively (for review, see Refs. 9 and 10). In addition, an SR␣ homologue has been identified in E. coli, designated FtsY (7, 11). Both P48 and FtsY (and their eukaryotic counterparts) are GTPases, and GTP binding and hydrolysis regulate the targeting cycle in a mechanism that has not yet been fully defined (12)(13)(14). In vivo overproduction of polytopic inner membrane proteins titrated out the limited amount of endogenous E. coli SRP (5), whereas depletion of essential SRP components affected membrane targeting of both polytopic and bitopic inner membrane proteins (3, 4).We have developed an in vitro cross-linking approach to dissect subsequent stages in SRP-mediated protein targeting. Short nascent inner m...

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“…The insertion mechanism in prokaryotes and eukaryotes is expected to be similar, because core components of the secretory translocase are conserved (7). In Escherichia coli, polytopic membrane protein insertion seems to require the secretory translocase (8)(9)(10) as well as other proteins needed for insertion in eukaryotes, such as the signal recognition particle (10,11). In addition, insertion of E. coli polytopic membrane proteins in vitro occurs cotranslationally (12)(13)(14).…”

mentioning

confidence: 99%

Ordered membrane insertion of an archaeal opsin in vivo

Dale

Angevine

Krebs

2000

Proc. Natl. Acad. Sci. U.S.A.

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Insertion of the Polytopic Membrane Protein MalF Is Dependent on the Bacterial Secretion Machinery

Cited by 97 publications

References 26 publications

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli

SecA Is Not Required for Signal Recognition Particle-mediated Targeting and Initial Membrane Insertion of a Nascent Inner Membrane Protein

Ordered membrane insertion of an archaeal opsin in vivo

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