FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1964517070> ?p ?o ?g. }

• W1964517070 endingPage "29706" @default.
• W1964517070 startingPage "29698" @default.
• W1964517070 abstract "SecA has been suggested to cycle on and off the cytoplasmic membrane of Escherichia coli during protein translocation. We have reconstituted 35S-SecA onto SecA-depleted membrane vesicles and followed the fate of the membrane-associated 35S-SecA during protein translocation. Some 35S-SecA was released from the membranes in a translocation-independent manner. However, a significant fraction of 35S-SecA remained on the membranes even after incubation with excess SecA. This fraction of 35S-SecA was shown to be integrated into the membrane and was active in protein translocation, indicating that SecA cycling on and off membrane is not required for protein translocation. Proteolysis experiments did not support the model of SecA insertion and deinsertion during protein translocation; instead, a major 48-kDa domain was found persistently embedded in the membrane regardless of translocation status. Thus, in addition to catalyzing ATP hydrolysis, certain domains of SecA probably play an important structural role in the translocation machinery, perhaps forming part of the protein-conducting channels. SecA has been suggested to cycle on and off the cytoplasmic membrane of Escherichia coli during protein translocation. We have reconstituted 35S-SecA onto SecA-depleted membrane vesicles and followed the fate of the membrane-associated 35S-SecA during protein translocation. Some 35S-SecA was released from the membranes in a translocation-independent manner. However, a significant fraction of 35S-SecA remained on the membranes even after incubation with excess SecA. This fraction of 35S-SecA was shown to be integrated into the membrane and was active in protein translocation, indicating that SecA cycling on and off membrane is not required for protein translocation. Proteolysis experiments did not support the model of SecA insertion and deinsertion during protein translocation; instead, a major 48-kDa domain was found persistently embedded in the membrane regardless of translocation status. Thus, in addition to catalyzing ATP hydrolysis, certain domains of SecA probably play an important structural role in the translocation machinery, perhaps forming part of the protein-conducting channels. INTRODUCTIONTranslocation of most periplasmic and outer membrane proteins across the cytoplasmic membrane of Escherichia coli occurs through the Sec-dependent pathway (1Fandl J. Tai P.C. J. Bioenerg. Biomembr. 1990; 22: 369-387Crossref PubMed Scopus (11) Google Scholar, 2Wickner W. Driessen A.J.M. Hartl F.-U. Annu. Rev. Biochem. 1991; 60: 101-124Crossref PubMed Scopus (338) Google Scholar), which consists of a distinct set of Sec proteins (3Schatz P.J. Beckwith J. Annu. Rev. Genet. 1990; 24: 215-248Crossref PubMed Scopus (270) Google Scholar, 4Bieker K.L. Phillips G.J. Silhavy T.J. J. Bioenerg. Biomembr. 1990; 22: 291-310Crossref PubMed Scopus (122) Google Scholar). SecA plays a pivotal role in protein translocation. It couples the energy of essential ATP hydrolysis to protein translocation (5Chen L. Tai P.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4384-4388Crossref PubMed Scopus (90) Google Scholar, 6Chen L. Tai P.C. Nature. 1987; 328: 164-166Crossref PubMed Scopus (27) Google Scholar, 7Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D.B. Wickner W. EMBO J. 1989; 8: 961-966Crossref PubMed Scopus (307) Google Scholar, 8Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (187) Google Scholar) and has been shown to be essential for protein translocation both in vivo (9Oliver D.B. Beckwith J. Cell. 1981; 25: 765-772Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 10Oliver D.B. Beckwith J. Cell. 1982; 30: 311-319Abstract Full Text PDF PubMed Scopus (213) Google Scholar) and in vitro (11Cabelli R.J. Chen L. Tai P.C. Oliver D.B. Cell. 1988; 55: 683-692Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 12Cunningham K. Lill R. Crooke E. Rice M. Moore K. Wickner W. Oliver D. EMBO J. 1989; 8: 955-959Crossref PubMed Scopus (140) Google Scholar, 13Kawasaki H. Matsuyama S. Sasaki S. Akita M. Mizushima S. FEBS Lett. 1989; 242: 431-434Crossref PubMed Scopus (46) Google Scholar, 14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar). SecA interacts with most components of the translocation machinery. It is believed that SecA binds to the membrane through interactions with SecYEG (15Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (445) Google Scholar, 16Douville K. Price A. Eichler J. Economou A. Wickner W. J. Biol. Chem. 1995; 270: 20106-20111Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and acidic phospholipids (17Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar, 18Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 19Ulbrandt N.D. London E. Oliver D.B. J. Biol. Chem. 1992; 267: 15184-15192Abstract Full Text PDF PubMed Google Scholar, 20Breukink E. Demel R.A. de Korte-Kool G. de Kruijff B. Biochemistry. 1992; 31: 1119-1124Crossref PubMed Scopus (157) Google Scholar, 21Breukink E. Nouwen N. van Raalte A. Mizushima S. Tommassen J. de Kruijff B. J. Biol. Chem. 1995; 270: 7902-7907Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), and interacts with precursor proteins by recognizing the positive charge at the NH2 terminus of the signal peptides (22Akita M. Sasaki S. Matsuyama S. Mizushima S. J. Biol. Chem. 1990; 265: 8164-8169Abstract Full Text PDF PubMed Google Scholar, 23Kimura E. Akita M. Matsuyama S. Mizushima S. J. Biol. Chem. 1991; 266: 6600-6606Abstract Full Text PDF PubMed Google Scholar). Binding of precursor proteins to the SecA subunit of the translocase stimulates the translocation ATPase activity of SecA (18Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar). Recently, it has been reported that SecA promotes protein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar) at SecYEG, and that this cycle is regulated by SecD/F (25Economou A. Poliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Abstract Full Text PDF PubMed Scopus (270) Google Scholar) and the ATP-binding amino terminus of SecA (26Rajapandi T. Oliver D. Mol. Microbiol. 1996; 20: 43-51Crossref PubMed Scopus (33) Google Scholar).While SecYEG are integral membrane proteins (27Brundage L. Fimmel C.J. Mizushima S. Wickner W. J. Biol. Chem. 1992; 267: 4166-4170Abstract Full Text PDF PubMed Google Scholar, 28Nishiyama K. Mizushima S. Tokuda H. EMBO J. 1993; 12: 3409-3415Crossref PubMed Scopus (129) Google Scholar), SecA is considered a peripheral membrane protein since it lacks any predicted transmembrane domains (29Schmidt M.G. Rollo E.E. Grodberg J. Oliver D.B. J. Bacteriol. 1988; 170: 3404-3414Crossref PubMed Scopus (131) Google Scholar), and was found in both cytosolic and membrane fractions (9Oliver D.B. Beckwith J. Cell. 1981; 25: 765-772Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 30Cabelli R.J. Dolan K.M. Qian L. Oliver D.B. J. Biol. Chem. 1991; 266: 24420-24427Abstract Full Text PDF PubMed Google Scholar). It is generally believed, therefore, that SecA cycles between the cytoplasm and the membrane during protein translocation (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 31Bieker-Brady K. Silhavy T.J. EMBO J. 1992; 11: 3165-3174Crossref PubMed Scopus (50) Google Scholar). Consistent with this notion, soluble SecA can restore translocation activity of membranes with depleted or inactivated SecA (11Cabelli R.J. Chen L. Tai P.C. Oliver D.B. Cell. 1988; 55: 683-692Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 12Cunningham K. Lill R. Crooke E. Rice M. Moore K. Wickner W. Oliver D. EMBO J. 1989; 8: 955-959Crossref PubMed Scopus (140) Google Scholar, 13Kawasaki H. Matsuyama S. Sasaki S. Akita M. Mizushima S. FEBS Lett. 1989; 242: 431-434Crossref PubMed Scopus (46) Google Scholar, 14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar) or impaired SecY (32Fandl J.P. Cabelli R. Oliver D. Tai P.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8953-8957Crossref PubMed Scopus (41) Google Scholar). On the other hand, extraction experiments with urea or Na2CO3 suggested that a significant fraction of SecA behaves like an integral membrane protein (30Cabelli R.J. Dolan K.M. Qian L. Oliver D.B. J. Biol. Chem. 1991; 266: 24420-24427Abstract Full Text PDF PubMed Google Scholar). 1L. Chen and P. C. Tai, unpublished results. Recently, Kim et al. (33Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar) reported that a strain harboring a plasmid containing the secD secF locus possesses SecA almost entirely as an integral membrane form and displays normal protein translocation as measured by rapid processing of preproteins in vivo (although it is possible that only a small fraction of SecA is active which accounts for the apparent “normal” translocation). Kim et al. (33Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar) suggested that the integral SecA is the catalytically active form in vivo and that, in this state, it might form a part of the reported protein-conducting channels (34Simon S.M. Blobel G. Cell. 1992; 69: 677-684Abstract Full Text PDF PubMed Scopus (170) Google Scholar). However, it is not clear what fraction of SecA is active and whether this integral SecA remains active and cycles off the membranes as suggested by Economou and Wickner (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar).Here, we present evidence that the integral SecA under the conditions used is indeed active and does not cycle off the membrane during protein translocation. We have determined the fate of the membrane-associated SecA during protein translocation, using membranes reconstituted from 35S-SecA and SecA-depleted inverted membrane vesicles. There were two forms of 35S-SecAs on the reconstituted membranes, loosely associated and integral. The loosely associated 35S-SecA can be replaced by excess nonradioactive SecA, and can be extracted by Na2CO3 or heparin. However, the release of this membrane-associated 35S-SecA is translocation-independent. On the other hand, a significant fraction of 35S-SecA remains on the membranes even after incubation with excess nonradioactive SecA. This fraction of 35S-SecA is integral, since it was found associated with membranes after flotation centrifugation and was also resistant to Na2CO3 extraction. After extraction with Na2CO3 or heparin, the 35S-SecA remaining on the reconstituted membranes was as active as the SecA remaining on native membranes. Proteolysis revealed that a 48-kDa domain is constantly embedded in the membranes regardless of protein translocation status. Together, these results indicate that a significant fraction of SecA is persistently embedded in the membrane and, as such, is active in protein translocation. Therefore, SecA cycling on and off membrane is not essential for protein translocation.DISCUSSIONWe have shown that there are two populations of SecA present on reconstituted membranes, loosely associated and tightly associated. Loosely associated SecA probably binds to membrane through ionic interaction between SecA and phospholipids. As a result, exchange of this loosely associated SecA is translocation-independent and such SecA could be stripped away from the membranes by the polyanionic heparin. Binding of SecA to the membrane is a spontaneous process, which can occur even on ice without ATP (Fig. 2A, and Ref. 15Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (445) Google Scholar). The tightly associated SecA might become inserted into the membrane. This possibly occurs through interactions with phospholipids, SecYEG (15Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (445) Google Scholar, 17Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar, 18Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 19Ulbrandt N.D. London E. Oliver D.B. J. Biol. Chem. 1992; 267: 15184-15192Abstract Full Text PDF PubMed Google Scholar, 20Breukink E. Demel R.A. de Korte-Kool G. de Kruijff B. Biochemistry. 1992; 31: 1119-1124Crossref PubMed Scopus (157) Google Scholar, 21Breukink E. Nouwen N. van Raalte A. Mizushima S. Tommassen J. de Kruijff B. J. Biol. Chem. 1995; 270: 7902-7907Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), or other proteins so that it becomes resistant to extraction by heparin or Na2CO3. Our data are consistent with evidence reported by Cabelli et al. (30Cabelli R.J. Dolan K.M. Qian L. Oliver D.B. J. Biol. Chem. 1991; 266: 24420-24427Abstract Full Text PDF PubMed Google Scholar), who showed that in membranes isolated by chromatography in buffer, one-third of the membrane-associated SecA could not be extracted by Na2CO3. In the current studies, the integral nature of this Na2CO3-resistant SecA was further confirmed and extended by both its resistance to replacement with excess nonradioactive SecA under translocation condition, and its association with membrane vesicles during flotation centrifugation. The amount of integral SecA is about 10 μg (50 pmol of dimers)/1 mg of membrane protein, similar to the amount of SecA present on membranes from the SecA+ parental CK1801 and wild type D10 strains. Most of SecA on these native membranes is also resistant to Na2CO3 and urea extraction,1 presumably because loosely associated SecA had previously been removed by the membrane preparation procedures, which consist of a series of density centrifugation in buffer containing EDTA (38Rhoads D.B. Tai P.C. Davis B.D. J. Bacteriol. 1984; 159: 63-70Crossref PubMed Google Scholar). It has been suggested that SecA cycles on and off the membrane during protein translocation (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 31Bieker-Brady K. Silhavy T.J. EMBO J. 1992; 11: 3165-3174Crossref PubMed Scopus (50) Google Scholar). However, this cycling model lacks direct experimental evidence. Our data show that a significant fraction of functional SecA is embedded in the membrane during protein translocation; therefore, SecA cycling on and off membrane is not essential in protein translocation.It appears highly likely that soluble SecA can bind to membranes and a fraction becomes integrated, but that, once integrated, SecA cannot be released into a soluble form. This integral SecA might be modified or rearranged to become permanently embedded in membranes. Several different forms of SecA in the cell have been reported (46Liebke H.H. J. Bacteriol. 1987; 169: 1174-1181Crossref PubMed Google Scholar). It has been reported (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 25Economou A. Poliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Abstract Full Text PDF PubMed Scopus (270) Google Scholar) that a 30-kDa domain of SecA becomes “protease-inaccessible” during protein translocation and that this 30-kDa fragment could become “protease-accessible” again by chasing it with excess nonradioactive SecA. Based on these data, the authors proposed a model in which membrane-associated SecA undergoes cycles of membrane insertion and deinsertion during protein translocation. However, our data do not support this model in our system. Instead, we found more than six SecA fragments after proteolysis of the reconstituted membranes (Fig. 8A). Similar proteolytic patterns were also observed when native membranes (detected by immunoblot) or membranes reconstituted from urea-washed membranes and 35S-SecA were used. 3X. Chen and Tai, unpublished observations. The 28-kDa fragment observed here may correspond to the 30-kDa fragment documented by Economou and Wickner (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar), since it increased in the presence of proOmpA, although the increase was much smaller (<3-fold) than reported. However, the 28-kDa fragment only accounts for a small fraction of total proteinase K-resistant SecA fragments on the membranes, and the majority of the 28-kDa fragment is in the supernatant regardless of translocation status (Fig. 8A). The differences in the proteolytic patterns might come from the different experimental conditions. Economou and Wickner (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar) reconstituted their membranes by incubating 125I-SecA with urea-washed membranes on ice in the absence of ATP, and collected membranes after proteolysis by trichloroacetic acid precipitation, which may include soluble protease-resistant SecA fragments. On the other hand, we reconstituted membranes by incubating 35S-SecA with SecA-depleted membranes at 30°C in the presence of ATP, and collected the membranes by centrifugation. However, even when the reconstitution, translocation, proteolysis, and membrane collection were performed following the described procedures (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar), all seven SecA fragments were still observed although the 28- and 25-kDa fragments became more prominent.3 The other difference is in the labeling of SecA. We used a uniform [35S]Met labeling in vivo, whereas Economou and Wickner (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar) labeled their SecA with 125I in vitro, which labels only surface-accessible tyrosine residues. Since most tyrosine residues will be embedded in the interior of soluble SecA, and the labeling efficiency is calculated as about 1% from the published data (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar), it is most likely that only 1 out of the total 25 tyrosine residues in the SecA was labeled. Therefore, only fragments containing this 125I-labeled tyrosine residue became detectable. The 66- and 48-kDa fragments may not contain the labeled tyrosine residues, thus, missing from their proteolytic patterns. However, we have no satisfactory explanation for the different observation on the 28-kDa/30-kDa fragments. It should be noted that in our hand, the 28- and 25-kDa fragments appear to be intrinsically resistant to proteinase K (1 mg/ml) upon the incubation of SecA with ATP in the presence of membranes.SecA has been proposed to function as a dimer (47Akita M. Shinkai A. Matsuyama S. Mizushima S. Biochem. Biophys. Res. Commun. 1991; 174: 211-216Crossref PubMed Scopus (97) Google Scholar, 48Driessen A.J.M. Biochemistry. 1993; 32: 13190-13197Crossref PubMed Scopus (130) Google Scholar). It is possible that integral SecA exists as a dimer in the membranes, since it is active in protein translocation. The presence of the constant 90-kDa and the 48-kDa SecA fragments (Fig. 8A) suggests that integral SecA is deeply embedded in the membrane. Thus, it is likely that there is a fraction of SecA dimer deeply embedded in the membranes that give rise to 90- and 48-kDa fragments upon protease digestion. These domains of SecA may form a part of the protein-conducting channels (33Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 34Simon S.M. Blobel G. Cell. 1992; 69: 677-684Abstract Full Text PDF PubMed Scopus (170) Google Scholar). Indeed, SecA has been shown to be the nearest neighbor of preproteins during translocation (49Joly J.C. Wickner W. EMBO J. 1993; 12: 255-263Crossref PubMed Scopus (147) Google Scholar). Therefore, SecA on the membranes may play a structural role as well as a catalytic role for protein translocation across the inner membrane. The structural role of the SecA may also provide an explanation for the findings that SecY-deficient membranes and SecE-deficient membranes are active in protein translocation in vitro (14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar, 50Watanabe M. Nicchitta C.V. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1960-1964Crossref PubMed Scopus (34) Google Scholar, 51Tai P.C. Lian J. Yu N.J. Fandl J. Xu H Vidugiriene J. Antonie van Leeuwenhoek. 1992; 61: 105-109Crossref PubMed Scopus (4) Google Scholar). 4J. P. Lian, Y. Yang, and P. C. Tai, manuscript in preparation. 5Y. Yang, N. Yu, and P. C. Tai, manuscript in preparation.Watanabe and Blobel (14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar) have reported that the urea- or heparin-extracted membrane vesicles, which contained only membrane-integral SecA, were fully active in protein translocation in the presence of F1-ATPase. In addition, they showed that reconstituted proteoliposomes from detergent extracts of heparin-extracted membrane vesicles containing only integral SecA but no F1- ATPase were fully active in protein translocation. The significance of this finding, however, has been questioned (33Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar), since the proteoliposomes also lack SecY, an “indispensable” component of the translocation machinery (52Nishiyama K. Kabuyama Y. Akimaru J. Matsuyama S. Tokuda H. Mizushima S. Biochim. Biophys. Acta. 1991; 1065: 89-97Crossref PubMed Scopus (38) Google Scholar). To address these concerns, we carried out heparin extraction with SecA reconstituted membranes and native membranes and confirmed that the extracted membranes are active (Fig. 7). The amount of SecY is not affected by the extraction and there is no F1-ATPase in the membranes. Thus, the translocation activity of these membranes is indeed due to the integral SecA. F1 forms complex with F0 on the wild-type membrane, blocking the proton flow through the F0 channel (53Brusilow W.S.A. Mol. Microbiol. 1993; 9: 419-424Crossref PubMed Scopus (17) Google Scholar). When F1 is removed, the membranes become permeable to protons (54Hasen S.M. Tsuchiya T. Rosen B.P. J. Bacteriol. 1978; 133: 108-113Crossref PubMed Google Scholar), possibly resulting in the collapse of proton motive force and loss of translocation activity (55Ernst F. Hoffschulte H.K. Thome-Kromer B. Swidersky U.E. Werner P.K. Müller M. J. Biol. Chem. 1994; 269: 12840-12845Abstract Full Text PDF PubMed Google Scholar). Restoration of translocation activity of urea-treated membranes by adding back soluble F1-ATPase (14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar) may occur through restoration of the proton motive force, which helps protein translocation under certain conditions (35Chen L. Tai P.C. J. Bacteriol. 1986; 167: 389-392Crossref PubMed Google Scholar, 42Schiebel E. Driessen A.J.M. Hartl F.-U. Wickner W. Cell. 1991; 64: 927-939Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 56Yamada H. Matsuyama S. Tokuda H. Mizushima S. J. Biol. Chem. 1989; 264: 18577-18581Abstract Full Text PDF PubMed Google Scholar, 57Shiozuka K. Tani K. Mizushima S. Tokuda H. J. Biol. Chem. 1990; 265: 18843-18847Abstract Full Text PDF PubMed Google Scholar). Since we used unc− membranes, the Na2CO3 and the heparin treatments do not disrupt the proton motive force due to the F1F0 ATPase. Therefore, F1-ATPase is not required for the translocation activity of the reconstituted membranes in our system. Watanabe and Blobel (14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar) also found that heparin-treated membranes containing less SecA are as active as the control membranes. However, we found that the translocation activity levels of heparin-treated membranes are lower than the control membranes, but are the same when normalized to the remaining SecA. This difference probably comes from the amount of precursor proteins used in the in vitro systems. The amount of SecA remaining on the membranes after heparin extraction may be sufficient for maximum translocation of the nascent precursor proteins whose amount is usually small. On the other hand, when purified precursors were used, there are usually more precursor molecules than SecA in the in vitro system. As a result, although adding soluble SecA to our reconstituted membranes did not enhance translocation of the nascent proOmpA (Fig. 1), it did increase the translocation efficiency when purified proOmpA was used (Fig. 4). Thus, our data support and extend these previous findings that integral SecA is active. Moreover, since each integral SecA molecule can support several cycles of OmpA translocation, SecA cycling on and off the membrane is clearly not an obligatory step for protein translocation.Whether or not cytosolic SecA plays an essential role in protein translocation is controversial. SecA has been reported to interact with the precursor proteins (22Akita M. Sasaki S. Matsuyama S. Mizushima S. J. Biol. Chem. 1990; 265: 8164-8169Abstract Full Text PDF PubMed Google Scholar, 23Kimura E. Akita M. Matsuyama S. Mizushima S. J. Biol. Chem. 1991; 266: 6600-6606Abstract Full Text PDF PubMed Google Scholar, 58Chun S.-Y. Randall L.L. J. Bacteriol. 1994; 176: 4197-4203Crossref PubMed Google Scholar). Additionally, soluble SecA/SecB complex has been detected in vivo and was shown to be involved in targeting precursor proteins to the membrane (59Hoffschulte H.K. Drees B. Müller M. J. Biol. Chem. 1994; 269: 12833-12839Abstract Full Text PDF PubMed Google Scholar). On the other hand, proOmpA or proOmpA/SecB complex has been shown to bind to SecA at membranes (15Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (445) Google Scholar) and the SecA/SecB cascade is not essential for protein translocation, since SecB only binds to a subset of preproteins. It is also known that soluble SecA is not required for translocation if sufficient SecA is bound to the membranes (11Cabelli R.J. Chen L. Tai P.C. Oliver D.B. Cell. 1988; 55: 683-692Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar). Recently, Kim et al. (33Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar) have shown that a strain with all its SecA on the membrane displayed normal translocation of preproteins in vivo. Our in vitro data shows that soluble SecA exchanges with the loosely bound SecA regardless of protein translocation status (Fig. 3, Fig. 4). What then, is the function of the cytosolic or free SecA? One possible function of cytosolic SecA is to shuttle the SecB/precursor from cytosol to membranes. Another known function for the cytosolic SecA is in the repression of its own synthesis (60Oliver D.B. Cabelli R.J. Jarosik G.P. J. Bioenerg. Biomembr. 1990; 22: 311-336Crossref PubMed Scopus (59) Google Scholar). SecA has been shown to compete with ribosomes for the ribosome binding site of its own mRNA (61Salavati R. Oliver D. RNA. 1995; 1: 745-753PubMed Google Scholar). It has been suggested that the accumulation of preproteins when translocation is impaired could be sensed by soluble SecA, leading to the derepression of SecA synthesis (62Rollo E.E. Oliver D.B. J. Bacteriol. 1988; 170: 3281-3282Crossref PubMed Google Scholar, 63Oliver D. Mol. Microbiol. 1993; 7: 159-165Crossref PubMed Scopus (75) Google Scholar). The increased soluble SecA levels, in turn, could compensate for the impaired Sec components, such as mutant SecY (32Fandl J.P. Cabelli R. Oliver D. Tai P.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8953-8957Crossref PubMed Scopus (41) Google Scholar, 62Rollo E.E. Oliver D.B. J. Bacteriol. 1988; 170: 3281-3282Crossref PubMed Google Scholar). Furthermore, SecA shares sequence homology with RNA helicase (64Koonin E.V. Gorbalenya A.E. FEBS Lett. 1992; 298: 6-8Crossref PubMed Scopus (47) Google Scholar) and binds to a variety of preprotein mRNAs (65Dolan K.M. Oliver D.B. J. Biol. Chem. 1991; 266: 23329-23333Abstract Full Text PDF PubMed Google Scholar). Therefore, soluble SecA may play a more important role in the regulation of protein expression. INTRODUCTIONTranslocation of most periplasmic and outer membrane proteins across the cytoplasmic membrane of Escherichia coli occurs through the Sec-dependent pathway (1Fandl J. Tai P.C. J. Bioenerg. Biomembr. 1990; 22: 369-387Crossref PubMed Scopus (11) Google Scholar, 2Wickner W. Driessen A.J.M. Hartl F.-U. Annu. Rev. Biochem. 1991; 60: 101-124Crossref PubMed Scopus (338) Google Scholar), which consists of a distinct set of Sec proteins (3Schatz P.J. Beckwith J. Annu. Rev. Genet. 1990; 24: 215-248Crossref PubMed Scopus (270) Google Scholar, 4Bieker K.L. Phillips G.J. Silhavy T.J. J. Bioenerg. Biomembr. 1990; 22: 291-310Crossref PubMed Scopus (122) Google Scholar). SecA plays a pivotal role in protein translocation. It couples the energy of essential ATP hydrolysis to protein translocation (5Chen L. Tai P.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4384-4388Crossref PubMed Scopus (90) Google Scholar, 6Chen L. Tai P.C. Nature. 1987; 328: 164-166Crossref PubMed Scopus (27) Google Scholar, 7Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D.B. Wickner W. EMBO J. 1989; 8: 961-966Crossref PubMed Scopus (307) Google Scholar, 8Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (187) Google Scholar) and has been shown to be essential for protein translocation both in vivo (9Oliver D.B. Beckwith J. Cell. 1981; 25: 765-772Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 10Oliver D.B. Beckwith J. Cell. 1982; 30: 311-319Abstract Full Text PDF PubMed Scopus (213) Google Scholar) and in vitro (11Cabelli R.J. Chen L. Tai P.C. Oliver D.B. Cell. 1988; 55: 683-692Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 12Cunningham K. Lill R. Crooke E. Rice M. Moore K. Wickner W. Oliver D. EMBO J. 1989; 8: 955-959Crossref PubMed Scopus (140) Google Scholar, 13Kawasaki H. Matsuyama S. Sasaki S. Akita M. Mizushima S. FEBS Lett. 1989; 242: 431-434Crossref PubMed Scopus (46) Google Scholar, 14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar). SecA interacts with most components of the translocation machinery. It is believed that SecA binds to the membrane through interactions with SecYEG (15Hartl F.-U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (445) Google Scholar, 16Douville K. Price A. Eichler J. Economou A. Wickner W. J. Biol. Chem. 1995; 270: 20106-20111Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and acidic phospholipids (17Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar, 18Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 19Ulbrandt N.D. London E. Oliver D.B. J. Biol. Chem. 1992; 267: 15184-15192Abstract Full Text PDF PubMed Google Scholar, 20Breukink E. Demel R.A. de Korte-Kool G. de Kruijff B. Biochemistry. 1992; 31: 1119-1124Crossref PubMed Scopus (157) Google Scholar, 21Breukink E. Nouwen N. van Raalte A. Mizushima S. Tommassen J. de Kruijff B. J. Biol. Chem. 1995; 270: 7902-7907Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), and interacts with precursor proteins by recognizing the positive charge at the NH2 terminus of the signal peptides (22Akita M. Sasaki S. Matsuyama S. Mizushima S. J. Biol. Chem. 1990; 265: 8164-8169Abstract Full Text PDF PubMed Google Scholar, 23Kimura E. Akita M. Matsuyama S. Mizushima S. J. Biol. Chem. 1991; 266: 6600-6606Abstract Full Text PDF PubMed Google Scholar). Binding of precursor proteins to the SecA subunit of the translocase stimulates the translocation ATPase activity of SecA (18Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar). Recently, it has been reported that SecA promotes protein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar) at SecYEG, and that this cycle is regulated by SecD/F (25Economou A. Poliano J.A. Beckwith J. Oliver D.B. Wickner W. Cell. 1995; 83: 1171-1181Abstract Full Text PDF PubMed Scopus (270) Google Scholar) and the ATP-binding amino terminus of SecA (26Rajapandi T. Oliver D. Mol. Microbiol. 1996; 20: 43-51Crossref PubMed Scopus (33) Google Scholar).While SecYEG are integral membrane proteins (27Brundage L. Fimmel C.J. Mizushima S. Wickner W. J. Biol. Chem. 1992; 267: 4166-4170Abstract Full Text PDF PubMed Google Scholar, 28Nishiyama K. Mizushima S. Tokuda H. EMBO J. 1993; 12: 3409-3415Crossref PubMed Scopus (129) Google Scholar), SecA is considered a peripheral membrane protein since it lacks any predicted transmembrane domains (29Schmidt M.G. Rollo E.E. Grodberg J. Oliver D.B. J. Bacteriol. 1988; 170: 3404-3414Crossref PubMed Scopus (131) Google Scholar), and was found in both cytosolic and membrane fractions (9Oliver D.B. Beckwith J. Cell. 1981; 25: 765-772Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 30Cabelli R.J. Dolan K.M. Qian L. Oliver D.B. J. Biol. Chem. 1991; 266: 24420-24427Abstract Full Text PDF PubMed Google Scholar). It is generally believed, therefore, that SecA cycles between the cytoplasm and the membrane during protein translocation (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 31Bieker-Brady K. Silhavy T.J. EMBO J. 1992; 11: 3165-3174Crossref PubMed Scopus (50) Google Scholar). Consistent with this notion, soluble SecA can restore translocation activity of membranes with depleted or inactivated SecA (11Cabelli R.J. Chen L. Tai P.C. Oliver D.B. Cell. 1988; 55: 683-692Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 12Cunningham K. Lill R. Crooke E. Rice M. Moore K. Wickner W. Oliver D. EMBO J. 1989; 8: 955-959Crossref PubMed Scopus (140) Google Scholar, 13Kawasaki H. Matsuyama S. Sasaki S. Akita M. Mizushima S. FEBS Lett. 1989; 242: 431-434Crossref PubMed Scopus (46) Google Scholar, 14Watanabe M. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9011-9015Crossref PubMed Scopus (38) Google Scholar) or impaired SecY (32Fandl J.P. Cabelli R. Oliver D. Tai P.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8953-8957Crossref PubMed Scopus (41) Google Scholar). On the other hand, extraction experiments with urea or Na2CO3 suggested that a significant fraction of SecA behaves like an integral membrane protein (30Cabelli R.J. Dolan K.M. Qian L. Oliver D.B. J. Biol. Chem. 1991; 266: 24420-24427Abstract Full Text PDF PubMed Google Scholar). 1L. Chen and P. C. Tai, unpublished results. Recently, Kim et al. (33Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar) reported that a strain harboring a plasmid containing the secD secF locus possesses SecA almost entirely as an integral membrane form and displays normal protein translocation as measured by rapid processing of preproteins in vivo (although it is possible that only a small fraction of SecA is active which accounts for the apparent “normal” translocation). Kim et al. (33Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar) suggested that the integral SecA is the catalytically active form in vivo and that, in this state, it might form a part of the reported protein-conducting channels (34Simon S.M. Blobel G. Cell. 1992; 69: 677-684Abstract Full Text PDF PubMed Scopus (170) Google Scholar). However, it is not clear what fraction of SecA is active and whether this integral SecA remains active and cycles off the membranes as suggested by Economou and Wickner (24Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar).Here, we present evidence that the integral SecA under the conditions used is indeed active and does not cycle off the membrane during protein translocation. We have determined the fate of the membrane-associated SecA during protein translocation, using membranes reconstituted from 35S-SecA and SecA-depleted inverted membrane vesicles. There were two forms of 35S-SecAs on the reconstituted membranes, loosely associated and integral. The loosely associated 35S-SecA can be replaced by excess nonradioactive SecA, and can be extracted by Na2CO3 or heparin. However, the release of this membrane-associated 35S-SecA is translocation-independent. On the other hand, a significant fraction of 35S-SecA remains on the membranes even after incubation with excess nonradioactive SecA. This fraction of 35S-SecA is integral, since it was found associated with membranes after flotation centrifugation and was also resistant to Na2CO3 extraction. After extraction with Na2CO3 or heparin, the 35S-SecA remaining on the reconstituted membranes was as active as the SecA remaining on native membranes. Proteolysis revealed that a 48-kDa domain is constantly embedded in the membranes regardless of protein translocation status. Together, these results indicate that a significant fraction of SecA is persistently embedded in the membrane and, as such, is active in protein translocation. Therefore, SecA cycling on and off membrane is not essential for protein translocation." @default.
• W1964517070 created "2016-06-24" @default.
• W1964517070 creator A5012396051 @default.
• W1964517070 creator A5016916322 @default.
• W1964517070 creator A5040052262 @default.
• W1964517070 date "1996-11-01" @default.
• W1964517070 modified "2023-09-27" @default.
• W1964517070 title "A Significant Fraction of Functional SecA Is Permanently Embedded in the Membrane" @default.
• W1964517070 cites W145694369 @default.
• W1964517070 cites W1480251791 @default.
• W1964517070 cites W1481018570 @default.
• W1964517070 cites W1488964125 @default.
• W1964517070 cites W1498240231 @default.
• W1964517070 cites W1511816051 @default.
• W1964517070 cites W1511994212 @default.
• W1964517070 cites W151834247 @default.
• W1964517070 cites W1520247815 @default.
• W1964517070 cites W1536997676 @default.
• W1964517070 cites W1572793754 @default.
• W1964517070 cites W1572886851 @default.
• W1964517070 cites W1576501498 @default.
• W1964517070 cites W1590719173 @default.
• W1964517070 cites W1602373369 @default.
• W1964517070 cites W1608494876 @default.
• W1964517070 cites W1610704689 @default.
• W1964517070 cites W1644489643 @default.
• W1964517070 cites W1664686765 @default.
• W1964517070 cites W1666626663 @default.
• W1964517070 cites W17566691 @default.
• W1964517070 cites W1895980260 @default.
• W1964517070 cites W1908462512 @default.
• W1964517070 cites W1946720111 @default.
• W1964517070 cites W1951366855 @default.
• W1964517070 cites W1967371046 @default.
• W1964517070 cites W1982019176 @default.
• W1964517070 cites W1992470956 @default.
• W1964517070 cites W1996635779 @default.
• W1964517070 cites W2004849849 @default.
• W1964517070 cites W2006195419 @default.
• W1964517070 cites W2008415333 @default.
• W1964517070 cites W2008828514 @default.
• W1964517070 cites W2022217156 @default.
• W1964517070 cites W2040338718 @default.
• W1964517070 cites W2041581829 @default.
• W1964517070 cites W2049434532 @default.
• W1964517070 cites W2052373858 @default.
• W1964517070 cites W2053211842 @default.
• W1964517070 cites W2055229289 @default.
• W1964517070 cites W2056130038 @default.
• W1964517070 cites W2056777456 @default.
• W1964517070 cites W2056891601 @default.
• W1964517070 cites W2066537459 @default.
• W1964517070 cites W2067411581 @default.
• W1964517070 cites W2073075045 @default.
• W1964517070 cites W2073725165 @default.
• W1964517070 cites W2080304004 @default.
• W1964517070 cites W2084334026 @default.
• W1964517070 cites W2093498871 @default.
• W1964517070 cites W2114561070 @default.
• W1964517070 cites W2122524469 @default.
• W1964517070 cites W2126106174 @default.
• W1964517070 cites W2145728481 @default.
• W1964517070 cites W2151739754 @default.
• W1964517070 cites W2151982140 @default.
• W1964517070 cites W2152737402 @default.
• W1964517070 cites W2154809686 @default.
• W1964517070 cites W2158291867 @default.
• W1964517070 cites W2160107586 @default.
• W1964517070 cites W2336712204 @default.
• W1964517070 cites W4240866489 @default.
• W1964517070 cites W4293247451 @default.
• W1964517070 cites W59314449 @default.
• W1964517070 doi "https://doi.org/10.1074/jbc.271.47.29698" @default.
• W1964517070 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8939903" @default.
• W1964517070 hasPublicationYear "1996" @default.
• W1964517070 type Work @default.
• W1964517070 sameAs 1964517070 @default.
• W1964517070 citedByCount "83" @default.
• W1964517070 countsByYear W19645170702012 @default.
• W1964517070 countsByYear W19645170702013 @default.
• W1964517070 countsByYear W19645170702014 @default.
• W1964517070 countsByYear W19645170702015 @default.
• W1964517070 countsByYear W19645170702017 @default.
• W1964517070 countsByYear W19645170702018 @default.
• W1964517070 countsByYear W19645170702019 @default.
• W1964517070 countsByYear W19645170702020 @default.
• W1964517070 countsByYear W19645170702021 @default.
• W1964517070 countsByYear W19645170702022 @default.
• W1964517070 countsByYear W19645170702023 @default.
• W1964517070 crossrefType "journal-article" @default.
• W1964517070 hasAuthorship W1964517070A5012396051 @default.
• W1964517070 hasAuthorship W1964517070A5016916322 @default.
• W1964517070 hasAuthorship W1964517070A5040052262 @default.
• W1964517070 hasBestOaLocation W19645170701 @default.
• W1964517070 hasConcept C12554922 @default.
• W1964517070 hasConcept C149629883 @default.
• W1964517070 hasConcept C185592680 @default.
• W1964517070 hasConcept C41625074 @default.

• next