共查询到20条相似文献,搜索用时 31 毫秒
1.
SecA suppresses the temperature-sensitive SecY24 defect in protein translocation in Escherichia coli membrane vesicles. 总被引:12,自引:2,他引:10
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J P Fandl R Cabelli D Oliver P C Tai 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(23):8953-8957
Genetic analysis of protein secretion in Escherichia coli has identified secY/prlA and secA as components of the secretory apparatus. We have examined the roles of the secY(prlA) gene product (an integral membrane protein) and the soluble secA gene product in translocation of OmpA and alkaline phosphatase precursors in an in vitro system. The protein translocation defect of the secY24 mutation was recently demonstrated in vitro as was its suppression by an S300 extract. We show here that the extract was essentially inactive in SecY24 suppression when SecA protein was removed from it by immunoaffinity chromatography. Furthermore, purified SecA protein suppressed the SecY24 defect. Preincubation of the inactivated SecY24 membrane vesicles either with S300 containing SecA or with purified SecA protein reconstituted the membranes and restored the translocation activity when assayed in the absence of additional soluble proteins. These results suggest that the SecY24 translocation defect is suppressed by SecA interacting, directly or indirectly, with SecY24 on the cytoplasmic membrane. 相似文献
2.
Residues essential for the function of SecE, a membrane component of the Escherichia coli secretion apparatus, are located in a conserved cytoplasmic region. 总被引:7,自引:1,他引:6
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
C K Murphy J Beckwith 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(7):2557-2561
Protein export in Escherichia coli is absolutely dependent on two integral membrane proteins, SecY and SecE. Previous deletion mutagenesis of the secE gene showed that only the third of three membrane-spanning segments and a portion of the second cytoplasmic region are necessary for its function in protein export. Here we further define the residues important for SecE function. Alignment of the SecE homologues of various eubacteria reveals that they all contain one membrane-spanning segment, compared with three in E. coli SecE, and that the most conserved region among them lies in their putative cytoplasmic amino termini; little homology exists in their membrane-spanning segments. The SecE homologue of the extreme thermophilic bacterium Thermotoga maritima was cloned and found to complement a deletion of secE in E. coli. Deletion or replacement of the cytoplasmic region of E. coli SecE eliminated SecE function, indicating that this sequence is essential for a functional secretion machinery. Mutant analysis suggests that the most important function of the third membrane-spanning segment is to maintain the proper topological arrangement of the conserved cytoplasmic domain. 相似文献
3.
Biochemical evidence for the secY24 defect in Escherichia coli protein translocation and its suppression by soluble cytoplasmic factors. 总被引:15,自引:7,他引:15
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J P Fandl P C Tai 《Proceedings of the National Academy of Sciences of the United States of America》1987,84(21):7448-7452
The secY (prlA) gene product is an integral membrane protein that has been identified genetically as one of the central components of the Escherichia coli protein translocation machinery. We have examined the effect of the secY24 (temperature-sensitive) mutation on the protein translocation activity of E. coli inverted membrane vesicles. Vesicles isolated from cells carrying this allele and grown at the nonpermissive temperature (42 degrees C) were less than 1% as active in translocation as vesicles isolated from an isogenic secY+ strain under the same conditions. Vesicles from the mutant strain grown at the permissive temperature (32 degrees C) were partially active, but those vesicles preincubated at 40 degrees C lost 90% of their activity. Moreover, the secY24 translocation defect on in vivo- or in vitro-inactivated vesicles was suppressed, or compensated, by an S300 soluble fraction from wild-type cells or from secY24 cells grown at nonpermissive temperature. The suppressing factor(s) was heat-labile and sensitive to proteinase K. These results provide biochemical evidence for the essential role of SecY in the translocation process and indicate that the translocation defect of SecY24 membranes can be compensated for by supplementing with additional soluble cytoplasmic proteins. 相似文献
4.
SecA protein is required for translocation of a model precursor protein into inverted vesicles of Escherichia coli plasma membrane. 总被引:7,自引:2,他引:5
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M Watanabe G Blobel 《Proceedings of the National Academy of Sciences of the United States of America》1993,90(19):9011-9015
We have investigated whether the SecA protein is required for in vitro translocation of a model presecretory protein into inverted vesicles (INV) of the Escherichia coli plasma membrane. Contrary to previous reports, we found that urea-extracted INV that contained only the membrane-integral form of SecA were fully translocation active. Proteoliposomes that were reconstituted from a detergent extract of INV did contain a full complement of membrane-integral SecA but < 1% of SecY. These proteoliposomes were fully translocation active. However, immunodepletion of > 90% of the SecA from the detergent extract yielded proteoliposomes that were translocation inactive. Addition of purified SecA to the SecA-depleted proteoliposomes restored translocation. The amounts of SecA required to saturate translocation activity were equivalent to those present as membrane-integral SecA in INV. These data indicate that SecA is necessary for protein translocation, and reinforce our previous conclusion that SecY is not required. Contrary to previous reports, we find that membrane-integral SecA is not irreversibly inactivated by 6 M urea and that membrane-integral SecA and SecY do not form a stoichiometric protein complex in the membrane. 相似文献
5.
A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex. 总被引:8,自引:1,他引:7
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
T Baba T Taura T Shimoike Y Akiyama T Yoshihisa K Ito 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(10):4539-4543
An approach to identifying the interaction site of multicomponent protein assembly has been applied to the membrane-bound SecY-SecE complex, which mediates protein export across the Escherichia coli cytoplasmic membrane. A dominant negative secY allele, secY-d1, inactivates SecY but preserves its ability to interact with SecE. Thus, the mutant protein sequesters SecE in an inactive complex. Second site mutations that disrupt the SecE binding site will suppress the export interference. We introduced insertion/deletion mutations that intragenically suppressed secY-d1. After eliminating knock-out mutations by virtue of the expression of a LacZ alpha sequence that had been attached to the C terminus, we obtained a striking clustering of mutations in cytoplasmic domain 4. On the basis of this result, the secY24 (Ts) substitution mutation in this domain was examined for its effects on interaction with SecE. It indeed suppressed secY-d1. Although the instability associated with excess SecY can be alleviated by overproduction of SecE, the secY24 mutant protein was not stabilized by SecE. The basal-level SecY24 protein was also destabilized at 42 degrees C. SecE was coimmunoprecipitated with SecY+ but not with the SecY24 protein. These results indicate that the secY24 mutation weakens SecY's interaction with SecE. Taken together, we propose that cytoplasmic domain 4 is important for the association between SecY and SecE. 相似文献
6.
Reconstitution of protein translocation from detergent-solubilized Escherichia coli inverted vesicles: PrlA protein-deficient vesicles efficiently translocate precursor proteins. 总被引:5,自引:3,他引:2
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M Watanabe C V Nicchitta G Blobel 《Proceedings of the National Academy of Sciences of the United States of America》1990,87(5):1960-1964
Proteoliposomes were reconstituted by detergent dialysis of a sodium cholate extract of inverted vesicles derived from Escherichia coli plasma membrane. The translocation of precursor proteins into reconstituted vesicles occurred at high efficiency and was SecB dependent. The protein composition of the reconstituted vesicles differed markedly from that of native vesicles. Immunoblot analysis of the sodium cholate extract and of the reconstituted vesicles indicated that PrlA (SecY) protein remained largely unsolubilized under the described conditions and was virtually absent from the reconstituted vesicles, suggesting that PrlA may not be required for in vitro translocation. 相似文献
7.
FtsH is required for proteolytic elimination of uncomplexed forms of SecY, an essential protein translocase subunit. 总被引:20,自引:0,他引:20
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A Kihara Y Akiyama K Ito 《Proceedings of the National Academy of Sciences of the United States of America》1995,92(10):4532-4536
When secY is overexpressed over secE or secE is underexpressed, a fraction of SecY protein is rapidly degraded in vivo. This proteolysis was unaffected in previously described protease-defective mutants examined. We found, however, that some mutations in ftsH, encoding a membrane protein that belongs to the AAA (ATPase associated with a variety of cellular activities) family, stabilized oversynthesized SecY. This stabilization was due to a loss of FtsH function, and overproduction of the wild-type FtsH protein accelerated the degradation. The ftsH mutations also suppressed, by alleviating proteolysis of an altered form of SecY, the temperature sensitivity of the secY24 mutation, which alters SecY such that its interaction with SecE is weakened and it is destabilized at 42 degrees C. We were able to isolate a number of additional mutants with decreased ftsH expression or with an altered form of FtsH using selection/screening based on suppression of secY24 and stabilization of oversynthesized SecY. These results indicate that FtsH is required for degradation of SecY. Overproduction of SecY in the ftsH mutant cells proved to deleteriously affect cell growth and protein export, suggesting that elimination of uncomplexed SecY is important for optimum protein translocation and for the integrity of the membrane. The primary role of FtsH is discussed in light of the quite pleiotropic mutational effects, which now include stabilization of uncomplexed SecY. 相似文献
8.
J C Joly M R Leonard W T Wickner 《Proceedings of the National Academy of Sciences of the United States of America》1994,91(11):4703-4707
SecY, SecE, and band 1 copurify as the SecY/E integral membrane domain of Escherichia coli preprotein translocase. To measure the in vivo association of these polypeptides and assay possible exchange, plasmid-borne secY and secE genes were placed under control of the ara regulon and fused to DNA encoding the influenza hemagglutinin epitope. Cells were incubated with [35S]methionine, grown for a "chase" period, and then induced with arabinose to express epitope-tagged, nonradioactive SecY and SecE. Both the wild-type and epitope-tagged polypeptides assembled into functional, heterotrimeric SecY/E complex. However, immunoprecipitation with antibody to the epitope tag did not cross-precipitate radiolabeled SecY or SecE. Thus, these subunits normally associate stably in vivo. 相似文献
9.
Detergent disruption of bacterial inner membranes and recovery of protein translocation activity. 总被引:2,自引:1,他引:1
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
K Cunningham W T Wickner 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(22):8673-8677
Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. We treated inner-membrane vesicles of Escherichia coli with mixtures of octyl beta-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure [35S]pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components. 相似文献
10.
Solubilization and functional reconstitution of the protein-translocation enzymes of Escherichia coli. 总被引:5,自引:3,他引:2
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A J Driessen W Wickner 《Proceedings of the National Academy of Sciences of the United States of America》1990,87(8):3107-3111
The SecY protein and other membrane proteins of Escherichia coli were solubilized by mixed micelles of n-octyl beta-D-glucopyranoside, phospholipids, and glycerol. Proteoliposomes formed from this extract by detergent dialysis supported energy-dependent translocation and processing of pro-OmpA. Translocation required ATP, SecY, and SecA and was stimulated by a proton-motive force. These results provide an important assay for the isolation and identification of membrane components involved in protein translocation. 相似文献
11.
An essential amino acid residue in the protein translocation channel revealed by targeted random mutagenesis of SecY
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Mori H Ito K 《Proceedings of the National Academy of Sciences of the United States of America》2001,98(9):5128-5133
The SecY/Sec61alpha family of membrane proteins are the central subunits of the putative protein translocation channel. We introduced random mutations into a segment of Escherichia coli SecY within its cytoplasmic domain 5, which was shown previously to be important for the SecA-dependent translocation activity. Mutations were classified into those retaining function and those gaining a dominant-interfering ability caused by a loss of function. These analyses showed that Arg-357, Pro-358, Gly-359, and Thr-362 are functionally important; Arg-357, conserved in almost all organisms, was identified as an indispensable residue. 相似文献
12.
R Kole S Altman 《Proceedings of the National Academy of Sciences of the United States of America》1979,76(8):3795-3799
RNase P preparations from Escherichia coli can be separated into RNA and protein by chromatography, in buffers containing 7 M urea, on Sephadex G-200, DEAE-Sephadex, or CM-Sephadex columns. Neither RNA nor protein components alone exhibits any RNase activity. RNase P activity can be reconstituted by mixing separated RNA and protein components in buffer containing 7M urea followed by dialysis of this mixture to remove the urea. Of several purified RNAs tried, only M2 RNA, the RNA species found in purified RNase P, is active in the reconstitution experiments. 相似文献
13.
Xiaoming Zhou Todd R. Graham 《Proceedings of the National Academy of Sciences of the United States of America》2009,106(39):16586-16591
Type-IV P-type ATPases (P4-ATPases) are putative phospholipid translocases, or flippases, that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. In addition, the activity of Drs2p, a P4-ATPase from Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi and endosomes, suggesting a role for phospholipid translocation in vesicle budding. Drs2p is necessary for translocation of a fluorescent phosphatidylserine analogue across purified Golgi membranes. However, a flippase activity has not been reconstituted with purified Drs2p or any other P4-ATPase, so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. Here, we show that Drs2p can catalyze phospholipid translocation directly through purification and reconstitution of this P4-ATPase into proteoliposomes. The noncatalytic subunit, Cdc50p, also was reconstituted in the proteoliposome, although at a substoichiometric concentration relative to Drs2p. In proteoliposomes containing Drs2p, a phosphatidylserine analogue was actively flipped across the liposome bilayer to the outer leaflet in the presence of Mg2+-ATP, whereas no activity toward the phosphatidylcholine or sphingomyelin analogues was observed. This flippase activity was mediated by Drs2p, because protein-free liposomes or proteoliposomes reconstituted with a catalytically inactive form of Drs2p showed no translocation activity. These data demonstrate for the first time the reconstitution of a flippase activity with a purified P4-ATPase. 相似文献
14.
Benedikt W. Bauer Tom A. Rapoport 《Proceedings of the National Academy of Sciences of the United States of America》2009,106(49):20800-20805
Many bacterial proteins, including most secretory proteins, are translocated across the plasma membrane by the interplay of the cytoplasmic SecA ATPase and a protein-conducting channel formed by the SecY complex. SecA catalyzes the sequential movement of polypeptide segments through the SecY channel. How SecA interacts with a broad range of polypeptide segments is unclear, but structural data raise the possibility that translocation substrates bind into a “clamp” of SecA. Here, we have used disulfide bridge cross-linking to test this hypothesis. To analyze polypeptide interactions of SecA during translocation, two cysteines were introduced into a translocation intermediate: one that cross-links to the SecY channel and the other one for cross-linking to a cysteine placed at various positions in SecA. Our results show that a translocating polypeptide is indeed captured inside SecA''s clamp and moves in an extended conformation through the clamp into the SecY channel. These results define the polypeptide path during SecA-mediated protein translocation and suggest a mechanism by which ATP hydrolysis by SecA is used to move a polypeptide chain through the SecY channel. 相似文献
15.
Reinhard C Schweikert M Wieland FT Nickel W 《Proceedings of the National Academy of Sciences of the United States of America》2003,100(14):8253-8257
Coat protein I (COPI)-coated transport vesicles mediate protein and lipid transport in the early secretory pathway. The basic machinery required for the formation of these transport intermediates has been elucidated based on the reconstitution of COPI-coated vesicle formation from chemically defined liposomes. In this experimental system, the coat components coatomer and GTP-bound ADP-ribosylation factor (ARF), as well as p23 as a membrane-bound receptor for COPI coat proteins, were shown to be both necessary and sufficient to promote COPI-coated vesicle formation. Based on biochemical and ultrastructural analyses, we now demonstrate that the catalytic domain of ARF-GTPase-activating protein (GAP) alone is sufficient to initiate uncoating of liposome-derived COPI-coated vesicles. By contrast, ARF-GAP activity is not required for COPI coat assembly and, therefore, does not seem to represent an essential coat component of COPI vesicles as suggested recently [Yang, J. S., Lee, S. Y., Gao, M., Bourgoin, S., Randazzo, P. A., et al. (2002) J. Cell Biol. 159, 69-78]. Thus, a complete round of COPI coat assembly and disassembly has been reconstituted with purified components defining the core machinery of COPI vesicle biogenesis. 相似文献
16.
Reconstitution of functional membrane-bound acetylcholine receptor from isolated Torpedo californica receptor protein and electroplax lipids. 总被引:7,自引:3,他引:4
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J M Gonzalez-Ros A Paraschos M Martinez-Carrion 《Proceedings of the National Academy of Sciences of the United States of America》1980,77(4):1796-1800
Purified acetylcholine receptor and total lipids, both extracted from Torpedo californica electroplax, were utilized to reconstitute chemically excitable membrane vesicles. Reconstitution was achieved by dialysis of the extraction detergent, octyl beta-D-glucoside from protein/lipid incubation mixtures. The reconstituted preparations could be fractionated by sucrose density gradient centrifugation and consisted of vesicular structures visible in electron micrographs. In addition, the reconstituted vesicles exhibited;the following properties characteristic of native receptor-enriched membranes: (i) an external distribution of alpha-bungarotoxin-binding sites, (ii) a time-dependent binding of alpha-bungarotoxin that is depressed by preincubation with the cholinergic agonist carbamoylcholine ("desensitization"), (iii) an ability to retain 22Na+ that is lost in the presence of detergents or gramicidin A, and (iv) a carbamoylcholine-induced acceleration of 22Na+ efflux that can be blocked by alpha-bungarotoxin. The purified acetylcholine receptor that was utilized in the reconstitution experiments apparently does not require other protein components for ligand recognition or ion translocation. 相似文献
17.
Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go. 总被引:2,自引:2,他引:2
下载免费PDF全文
![点击此处可从《Proceedings of the National Academy of Sciences of the United States of America》网站下载免费的PDF全文](/ch/ext_images/free.gif)
H Ueda H Harada M Nozaki T Katada M Ui M Satoh H Takagi 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(18):7013-7017
Reconstitution of purified mu opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. mu opioid receptors were purified by 6-succinylmorphine AF-AminoTOYOPEARL 650M affinity chromatography and by PBE isoelectric chromatography. The purified mu opioid receptor (pI 5.6) migrated as a single Mr 58,000 polypeptide by NaDodSO4/PAGE, a value identical to that obtained by affinity cross-linking purified mu receptors. When purified mu receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone (a mu opioid antagonist) binding by [D-Ala2,MePhe4,Gly-ol5]enkephalin (a mu opioid agonist) was increased 215-fold; this increase was abolished by adding 100 microM (guanosine 5'-[gamma-thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5'-[gamma-thio]triphosphate were observed with Go, the G protein of unknown function, but not with the v-Ki-ras protein p21. In reconstituted preparations with Gi or Go, neither [D-Pen2,D-Pen5]enkephalin (a delta opioid agonist; where Pen is penicillamine) nor U-69,593 (a kappa opioid agonist) showed displacement of the [3H]naloxone binding. In addition, the mu agonist stimulated both [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding (in exchange for GDP) and the low-Km GTPase in such reconstituted preparations, with Gi and Go but not with the v-Ki-ras protein p21, in a naloxone-reversible manner. The stoichiometry was such that the stimulation of 1 mol of mu receptor led to the binding of [3H]guanosine 5'-[beta,gamma-imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified mu opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles. 相似文献
18.
Akopyan K Edgren T Wang-Edgren H Rosqvist R Fahlgren A Wolf-Watz H Fallman M 《Proceedings of the National Academy of Sciences of the United States of America》2011,108(4):1639-1644
Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model. 相似文献
19.
Reconstitution of translocation-competent membrane vesicles from detergent-solubilized dog pancreas rough microsomes. 总被引:6,自引:0,他引:6
Y H Yu Y Y Zhang D D Sabatini G Kreibich 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(24):9931-9935
Dog pancreas rough microsomes were solubilized in 1% octyl beta-glucoside, and membrane vesicles were reconstituted by slow 30-fold dilution with a buffer of low ionic strength. Asymmetric assembly of the membranes occurred during reconstitution since the vesicles formed contained ribosomes bound only to the vesicular outer surfaces. The reconstituted vesicles were similar in protein composition to native rough microsomes, although these vesicles were largely devoid of luminal-content proteins. These reconstituted vesicles could translocate and process nascent secretory (human placental lactogen) and membrane proteins (influenza hemagglutinin and rat liver ribophorin I) synthesized in cell-free translation systems programmed with the corresponding mRNAs. Signal cleavage and N-glycosylation only occurred when the reconstituted membranes were present during translation, providing evidence that the translocation apparatus was asymmetrically assembled into the reconstituted membranes. When a supernatant lacking ribosomes and particles greater than 50S from centrifuging the detergent-solubilized microsomes at high speed was used for reconstitution, smooth-surfaced membrane vesicles were obtained that, except for the absence of ribosomal proteins, were similar in protein composition to that of the reconstituted vesicles from total solubilized rough microsomes. The reconstituted smooth-surfaced vesicles, however, were totally inactive in cotranslational processing and translocation of nascent polypeptides. These findings suggest that ribosomes and/or large macromolecular complexes, not dissociated under our solubilization conditions, are essential for in vitro assembly of a functional translocation apparatus. 相似文献
20.
The study of the 9S, untransformed state of steroid receptors has led to the discovery of a multiprotein chaperone system that assembles heterocomplexes between hsp90 and a variety of proteins involved in signal transduction. Using the formation of glucocorticoid receptor (GR)-hsp90 heterocomplexes as a model, we have reconstituted a fully functional heterocomplex assembly system from purified components. The basic assembly system requires four proteins-hsp90, hsp70, p60/Hop and hsp40-to assemble GR-hsp90 heterocomplexes, which are then stabilized by the hsp90-interacting protein p23. The four proteins can self-assemble into an hsp90-p60/Hop-hsp70-hsp40 complex that we call a foldosome. Foldosomes isolated from reticulocyte lysate or formed from purified proteins open up a steroid-binding pocket to create a high-affinity steroid-binding state of the GR. We describe here the systematic reconstitution of the hsp90-based chaperone machinery and develop a model of the receptor-hsp90 heterocomplex assembly mechanism. 相似文献