Syd, a SecY-interacting Protein, Excludes SecA from the SecYE Complex with an Altered SecY24 Subunit

Matsuo, Eiichi; Mori, Hiroyuki; Shimoike, Takashi; Ito, Koreaki

doi:10.1074/jbc.273.30.18835

Cited by 26 publications

(26 citation statements)

References 34 publications

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“…The insertion that is observed in response to a nonhydrolyzable analog of ATP still requires the integrity of the SecYE channel (18), although it is a distinct reaction in that it can be affected differentially by the secY205 mutation (17). Since the latter mode of insertion as well was stimulated by the SecY125 alteration, the secY125 mutation is likely to affect a step that is common between the productive and idling reactions (17) of SecA.…”

Section: Discussionmentioning

confidence: 99%

A Mutation in secY That Causes Enhanced SecA Insertion and Impaired Late Functions in Protein Translocation

et al. 2000

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A cold-sensitive secY mutant (secY125) with an amino acid substitution in the first periplasmic domain causes in vivo retardation of protein export. Inverted membrane vesicles prepared from this mutant were as active as the wild-type membrane vesicles in translocation of a minute amount of radioactive preprotein. The mutant membrane also allowed enhanced insertion of SecA, and this SecA insertion was dependent on the SecD and SecF functions. These and other observations suggested that the early events in translocation, such as SecA-dependent insertion of the signal sequence region, is actually enhanced by the SecY125 alteration. In contrast, since the mutant membrane vesicles had decreased capacity to translocate chemical quantity of pro-OmpA and since they were readily inactivated by pretreatment of the vesicles under the conditions in which a pro-OmpA translocation intermediate once accumulated, the late translocation functions appear to be impaired. We conclude that this periplasmic secY mutation causes unbalanced early and late functions in translocation, compromising the translocase's ability to catalyze multiple rounds of reactions.

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Section: Discussionmentioning

confidence: 99%

A Mutation in secY That Causes Enhanced SecA Insertion and Impaired Late Functions in Protein Translocation

et al. 2000

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“…secY24 mutant cells are extremely sensitive to overproduction of Syd, a SecY-interacting protein. In the presence of excess amounts of Syd, the mutationally altered SecY24-SecE channel seems to be destroyed with respect to the high-affinity binding of SecA, and this phenotype can be observed at any temperature (18,19,28).…”

Section: Complementation Abilities Of Secy Variants With C-terminal Tmentioning

confidence: 99%

Roles of the C-Terminal End of SecY in Protein Translocation and Viability of Escherichia coli

2002

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SecY, a central component of the membrane-embedded sector of protein translocase, contains six cytosolic domains. Here, we examined the importance of the C-terminal cytosolic region of SecY by systematically shortening the C-terminal end and examining the functional consequences of these mutations in vivo and in vitro. It was indicated that the C-terminal five residues are dispensable without any appreciable functional defects in SecY. Mutants missing the C-terminal six to seven residues were partially compromised, especially at low temperature or in the absence of SecG. In vitro analyses indicated that the initial phase of the translocation reaction, in which the signal sequence region of the preprotein is inserted into the membrane, was affected by the lack of the C-terminal residues. SecA binding was normal, but SecA insertion in response to ATP and a preprotein was impaired. It is suggested that the C-terminal SecY residues are required for SecA-dependent translocation initiation.Translocation of newly synthesized Escherichia coli proteins from the cytosol across the cytoplasmic membrane is facilitated by concerted actions of the SecA ATPase and the SecYEG integral membrane protein complex (for reviews, see references 14 and 22). Translocation is initiated by membrane insertion of the signal peptide and the following mature segment, leading to periplasmic exposure of the leader peptidase cleavage site. While initiation is triggered by ATP binding to SecA, continuation of translocation requires repeated hydrolysis cycles of ATP and/or the proton motive force (PMF) across the membrane. These processes appear to be closely related to the insertion/deinsertion cycle of SecA (5, 6). In this cycle, an ATP-and preprotein-dependent conformational change of the membrane-bound form of SecA results in its insertion into the membrane, which is followed by ATP hydrolysis-dependent release of the preprotein and deinsertion of SecA. These dynamic functions of SecA occur in its close interaction with the SecYEG components (5,15,22).SecY is the central subunit of the SecYEG complex and contains 10 transmembrane segments (TM1 to TM10), five periplasmic regions (P1 to P5), and six cytosolic domains (C1 to C6). The SecYEG complex is believed to provide a channellike pathway for protein translocation, probably by its oligomeric superassembly (4, 13). We isolated and characterized a number of cold-sensitive secY mutants in which protein export and cell growth were retarded at 20°C (22). In vivo and in vitro studies using these and the dominant-negative secY mutations suggested that the C-terminal cytosolic domains, C5 and C6, are particularly important for SecY functions (21, 27). Mutations in these domains significantly compromise the SecA-activating functions of SecY (33). In particular, a mutation (secY205) in C6 that changed Tyr429 (the 15th residue from the C terminus) to Asp was found to impair the SecA insertion reaction (15).To further assess the importance of the C-terminal residues of SecY, we carried out systematic stu...

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“…Preparation of Biochemical Materials-Inverted membrane vesicles (IMVs) were prepared from the SecYEG-overproducing strain (30,31). ProOmpA (C290G) was purified from its overproducing strain by the published procedures (29).…”

Section: Methodsmentioning

confidence: 99%

The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

Mori

Ito

2006

Journal of Biological Chemistry

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SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.

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Syd, a SecY-interacting Protein, Excludes SecA from the SecYE Complex with an Altered SecY24 Subunit

Cited by 26 publications

References 34 publications

A Mutation in secY That Causes Enhanced SecA Insertion and Impaired Late Functions in Protein Translocation

A Mutation in secY That Causes Enhanced SecA Insertion and Impaired Late Functions in Protein Translocation

Roles of the C-Terminal End of SecY in Protein Translocation and Viability of Escherichia coli

The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

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