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PubMed1995

Assembly of F0 sector of Escherichia coli H+ ATP synthase. Interdependence of subunit insertion into the membrane.

The F0 sector of the Escherichia coli H+ transporting ATP synthase is composed of a complex of three subunits, each of which traverses the inner membrane. We have studied the interdependence of subunit insertion into the membrane in a series of chromosomal mutants in which the primary mutation prevented insertion of one of the F0 subunits. Subunit insertion was assessed using Western blots of mutant membrane preparations. Subunit b and subunit c were found to insert into the membrane independently of the other two F0 subunits. On the other hand, subunit a was not inserted into membranes that lacked either subunit b or subunit c. The conclusion that subunit a insertion is dependent upon the co-insertion of subunits b and c differs from the conclusion of several studies, where subunits were expressed from multicopy plasmids.

Cell Membrane↗
PubMed1997

Light-harvesting chlorophyll a/b-binding protein stably inserts into etioplast membranes supplemented with Zn-pheophytin a/b.

Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.

Chlorophyll↗
PubMed1997

Topology mapping of the amino-terminal half of multidrug resistance-associated protein by epitope insertion and immunofluorescence.

The multidrug resistance-associated protein (MRP) is an integral membrane protein that causes multidrug resistance when overexpressed in mammalian cells. Within the ATP-binding cassette superfamily, MRP belongs to a subgroup of structurally and functionally related proteins that includes the yeast cadmium factor 1 and yeast oligomycin resistance I proteins, and the mammalian sulfonylurea receptors SUR1 and SUR2. Hydropathy analysis of these proteins predicts a unique membrane-associated region at the amino terminus followed by a structural unit composed of 12 transmembrane (TM) domains and two nucleotide-binding domains that is characteristic of eukaryotic ATP-binding cassette transporters. The topology of the membrane-associated regions of MRP remains largely unknown and was investigated. Small hemagglutinin epitopes (YPYDVPDYAS) were inserted in predicted hydrophilic segments of the membrane-associated regions from the amino-terminal half of MRP and these proteins were expressed in HeLa cells, and tested for their capacity to confer etoposide resistance. The polarity of the inserted tags with respect to plasma membrane was then deduced by immunofluorescence in intact and permeabilized cells. Insertion of epitopes at positions 4, 163, 271, 574, and 653 produced functional proteins while insertions at positions 127, 417, 461, and 529 abrogated the capacity of MRP to confer drug resistance. Epitopes inserted at positions 4, 163, and 574 were localized extracellularly, whereas those inserted at positions 271 and 653 revealed an intracellular location. Although a single epitope inserted at position 461 was compatible with MRP function, it was inaccessible to the anti-epitope antibody and two copies of the tag at that site abrogated MRP function. These results indicate that the amino terminus of MRP is extracellular, while the linker segment joining the first and second membrane-associated regions is intracellular as is the first nucleotide-binding domain. Our findings are therefore consistent with a topological model of MRP that contains 5 TM segments in the first membrane-associated region and 6 TM segments in the second membrane region.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Multivalent mechanism of membrane insertion by the FYVE domain.

Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with PtdIns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids.

Binding Sites↗
PubMed2004

Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers.

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.

Adenosine Triphosphate↗
PubMed2004

Sec/SRP requirements and energetics of membrane insertion of subunits a, b, and c of the Escherichia coli F1F0 ATP synthase.

Previously, the role of YidC in the membrane protein biogenesis of the F(0) sector of the Escherichia coli F(1)F(0) ATP synthase was investigated. Whereas subunits a and c of the F(1)F(0) ATP synthase were strictly dependent on YidC for membrane insertion, subunit b required YidC for efficient insertion (Yi, L., Jiang, F., Chen, M., Cain, B., Bolhuis, A., and Dalbey, R. E. (2003) Biochemistry 42, 10537-10544). In this paper, we investigated other protein components and energetics that are required in the membrane protein assembly of the F(0) sector subunits. We show here that the Sec translocase and the signal recognition particle (SRP) pathway are required for membrane insertion of subunits a and b. In contrast, subunit c required neither the Sec machinery nor the SRP pathway for insertion. While the proton motive force was not required for insertion of subunits b and c, it was required for translocation of the negatively charged periplasmic NH(2)-terminal tail of subunit a, whereas periplasmic loop 2 of subunit a could insert in a proton motive force-independent manner. Taken together, the in vivo data suggest that subunits a and b are inserted by the Sec/SRP pathway with the help of YidC, and subunit c is integrated into the membrane by the novel YidC pathway.

Adenosine Triphosphatases↗
PubMed2006

The properties of the positively charged loop region in PSI-G are essential for its "spontaneous" insertion into thylakoids and rapid assembly into the photosystem I complex.

The PSI-G subunit of photosystem I (PSI) is an 11-kDa membrane protein that plays an important role in electron transport between plastocyanin and PSI and is involved in the stability of the PSI complex. Within the complex, the PSI-G subunit is bound to PSI-B and is in contact with Lhca1. PSI-G has two transmembrane spans connected by a positively charged stromal loop. The loop is inaccessible to proteases, indicating a tightly bound location within the PSI complex. Here, we have studied the insertion mechanism and assembly of PSI-G. We show that the protein inserts into thylakoids by a direct or "spontaneous" pathway that does not involve the activities of any known chloroplast protein-targeting machinery. Surprisingly, the positively charged stromal loop region plays a major role in this process. Mutagenesis or deletions within this region almost invariably lead to a marked lowering of insertion efficiency, strongly indicating a critical role for the loop in the organization of the transmembrane regions prior to or during membrane insertion. Finally, we have examined the assembly of newly inserted PSI-G into the PSI complex, since very little is known of the assembly pathway for this large multimeric complex. Interestingly, we find that inserted PSI-G can be found within the full PSI complex within the import assay time frame after insertion into thylakoids, strongly suggesting that PSI-G normally associates at the end of the assembly process. This is consistent with its location on the periphery of the complex.

Adenosine Triphosphate↗
PubMed2006

New insights from the structure-function analysis of the catalytic region of human platelet phosphodiesterase 3A: a role for the unique 44-amino acid insert.

Human phosphodiesterase 3A (PDE3A) degrades cAMP, the major inhibitor of platelet function, thus potentiating platelet function. Of the 11 human PDEs, only PDE3A and 3B have 44-amino acid inserts in the catalytic domain. Their function is not clear. Incubating Sp-adenosine-3',5'-cyclic-S-(4-bromo-2,3-di-oxobutyl) monophosphorothioate (Sp-cAMPS-BDB) with PDE3A irreversibly inactivates the enzyme. High pressure liquid chromatography (HPLC) analysis of a tryptic digest yielded an octapeptide within the insert of PDE3A ((K)T(806)YNVTDDK(813)), suggesting that a substrate-binding site exists within the insert. Because Sp-cAMPS-BDB reacts with nucleophilic residues, mutants Y807A, D811A, and D812A were produced. Sp-cAMPS-BDB inactivates D811A and D812A but not Y807A. A docking model showed that Tyr(807) is 3.3 angstroms from the reactive carbon, whereas Asp(811) and Asp(812) are >15 angstroms away from Sp-cAMPS-BDB. Y807A has an altered K(m) but no change in k(cat). Activity of wild type but not Y807A is inhibited by an anti-insert antibody. These data suggest that Tyr(807) is modified by Sp-cAMPS-BDB and involved in substrate binding. Because the homologous amino acid in PDE3B is Cys(792), we prepared the mutant Y807C and found that its K(m) and k(cat) were similar to the wild type. Moreover, Sp-cAMPS-BDB irreversibly inactivates Y807C with similar kinetics to wild type, suggesting that the tyrosine may, like the cysteine, serve as a H donor. Kinetic analyses of nine additional insert mutants reveal that H782A, T810A, Y814A, and C816S exhibit an altered k(cat) but not K(m), indicating that catalysis is modulated. We document a new functional role for the insert in which substrate binding may produce a conformational change. This change would allow the substrate to bind to Tyr(807) and other amino acids in the insert to interact with residues important for catalysis in the active site cleft.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Cutaneous microdialysis in the rat: insertion trauma studied by ultrasound imaging.

Microdialysis is a method by which compounds can be studied in the extracellular space in skin, in vivo. The microdialysis probe is inserted in the dermis using a guide cannula. It is expected that intradermal oedema associated with insertion trauma as well as the probe depth can influence the results of microdialysis studies. The aim of the present study was to assess the effects of insertion trauma and, additionally, the probe depth by ultrasound. High-frequency (20 MHz) ultrasound is a noninvasive method for measuring the thickness and echostructure of skin. Hairless rats were anaesthetized with either halothane (n = 7) or pentobarbital sodium (n = 6). The insertion of the microdialysis probe resulted in a 38% relative increase in skin thickness. At 120 min the skin thickness had not reached the pre-insertion value. Thus, significant skin thickening, representing traumatic oedema, developed due to insertion of the microdialysis probe in the skin. The microdialysis probe could be inserted reproducibly in the lower dermis.

Administration, Cutaneous↗
PubMed2006

Effect of deep insertion of the cochlear implant electrode array on pitch estimation and speech perception.

CONCLUSION: Deeply inserted electrodes offer the possibility that apical stimulation may improve speech performances. Therefore, deep insertion is reasonable and should be performed in patients with profound or total hearing loss. OBJECTIVES: To evaluate the importance of insertion depth beyond 25 mm in a group of cochlear implant patients with deeply inserted electrodes up to 32 mm. PATIENTS AND METHODS: In the first part of the study patients were asked to perform a pitch estimation for channels across the whole length of the electrode array. We evaluated whether pitch discrimination was possible along the whole cochlea and especially in its apical part. Then, the audiological performances of 10 patients were tested in 5 conditions, in which we artificially varied the insertion depth in each patient by activating and deactivating channels. The patients were tested immediately in the new condition to avoid adaptation. RESULTS: The results showed that activating the electrodes in the uppermost region of the cochlea improves speech perception significantly. Furthermore it could be demonstrated that the pitch perceived in the cochlea with electrical stimulation decreases with increasing insertion depth along the whole length of deeply inserted electrode arrays.

Adult↗
PubMedDate not supplied

Endotracheal intubation and esophageal tracheal Combitube insertion by regular ambulance attendants: a comparative trial.

OBJECTIVE: Recent cardiac arrest resuscitation guidelines have recommended the esophageal tracheal Combitube (ETC) as an advanced airway management alternative for individuals who infrequently perform endotracheal intubation (ETI). This study attempted to analyze basic (nonparamedic) ambulance attendant success rates at ETI and ETC insertion as well as their continuing skill competency over time and whether ongoing practice on mannequins improved skill performance. METHODS: Three hundred fifty-seven adult patients in cardiorespiratory arrest were treated by 81 basic ambulance attendants. Original study design called for the analysis of two treatment options in three patient groups: ETC insertion, ETI insertion with mannequin practice (ETI-MP), and ETI insertion without mannequin practice (ETI-NMP). The main outcome measures were:successful insertion and ventilation with ETC or ETI, assessed by receiving physicians; and differences in successful insertion/ventilation between the MP and NMP groups. RESULTS: Successful insertion (intent-to-treat) for the ETI-NMP group was 70 of 111 (63%; 95% confidence interval [CI], 54-73%); ETI-MP success was 105 of 139 (76%; 95% CI, 67-84%); ETC-NMP success was 26 of 42 (62%; 95% CI, 49-75%); and ETC-MP success was 36 of 53 (68%; 95% CI, 54-82%). Continuing mannequin practice appeared to improve ETI success (as-treated): MP 75% versus NMP 61% (odds ratio, 2.1; 95% CI, 1.11-3.94). CONCLUSIONS: There were similar rates of successful insertion/ventilation with the ETC and ETI. ETI insertion success was lower without mannequin practice. ETI skill erosion was partially mitigated by additional field experience.

Adult↗
PubMed1993

In vitro insertion of the 22-kD peroxisomal membrane protein into isolated rat liver peroxisomes.

The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.

Animals↗
PubMed1979

Synthesis, insertion into the plasma membrane, and turnover of alpha-bungarotoxin receptors in chick sympathetic neurons.

alpha-Bungarotoxin was used to identify an integral membrane protein in the plasma membrane of chick sympathetic neurons. The synthesis, insertion into the plasma membrane, and turnover of the alpha-bungarotoxin receptor were studied using isotopically labeled amino acids (2H, 13C, 15N) to directly label receptor molecules. Neurons incubated in medium containing dense amino acids continued to insert unlabeled receptors from a pool of previously synthesized molecules for 2 h. Density-labeled receptors began to appear in the plasma membrane after this 2-h period. Synthesis of receptors, but not insertion into the surface, was blocked by cycloheximide (100 microgram/ml). Neither colchicine (0.05 microgram/ml) of actinomycin D (5 microgram/ml) has any effect on alpha-bungarotoxin receptor synthesis or insertion. Autoradiographic studied revealed that receptors occur on growth cones, axons, and cell bodies of single neurons and explanted ganglia. The rate of insertion of newly synthesized receptors into the plasma membrane of axons extending from explanted sympathetic ganglia was approximately the same as that into the cell body portion of the ganglion. Cytochalasin B (2 microgram/ml) rapidly distrupted growth cones but had no effect on receptor insertion. These experiments suggested that the growth cone is not the sole or even the primary site for insertion of this membrane protein. The kinetics of turnover of the alpha-bungarotoxin receptor were a first-order exponential with t 1/2 = 11 h. Neurons that had their surface receptors labeled with 125I-alpha-bungarotoxin produced [125I]iodotyrosine. This process was inhibited by low temperature (23 degrees C) and also by a metabolic inhibitor. This is interpreted as evidence that receptors turn over by a mechanism in which they are internalized and then proteolytically degraded.

Amino Acids↗
PubMed2006

Durability of working channel in flexible ureteroscopes when inserting ureteroscopic devices.

PURPOSE: We studied the physical damage to the working channel of flexible ureteroscopes caused by insertion of various accessories. A procedure was developed to avoid channel damage. MATERIALS AND METHODS: An experimental model representing a flexible ureteroscope was prepared, and damage to its working channel was evaluated by inserting instruments through it. Deflection angles of the channel were changed from 0 degrees to 120 degrees, and each device was inserted and removed 100 times for each selected angle of the channel. Any induced pinholes were identified by an air-leak test. Also, the inside of the channel was inspected with an extremely fine fiberscope. RESULTS: Insertions of 3F biopsy forceps or a 2.4F Nitinol stone-retrieval device caused only slight damage to the model channel, even when the deflection angle was 120 degrees. However, the tips of 200- or 250-microm holmium laser fibers shaved the inner surface of the channel at 60 degrees of deflection, and at 120 degrees, the laser fiber either penetrated the channel or could not be advanced because of resistance by the channel wall. When the laser fiber was inserted within a protective tube, the channel was never damaged, even when the deflection angle was 120 degrees. CONCLUSIONS: When devices are inserted into the working channel of a flexible ureteroscope, damage to the wall depends on the kind of device and deflection angle. Harm could be avoided by inserting the devices, especially laser probes, within a protective tube.

Equipment Design↗
PubMed1997

Tn916 transposition in Haemophilus influenzae Rd: preferential insertion into noncoding DNA.

The availability of completely sequenced genomes has created an opportunity for high throughput mutational studies. Using the conjugative transposon Tn916, a pilot project was initiated to determine the efficiency of gene disruption in the first completely sequenced bacterium, Haemophilus influenzae Rd strain KW20. DNA was isolated from Tn916-mutagenized cells, and the point of transposon insertion was determined by inverse PCR, DNA sequencing, and mapping to the wild-type genome sequence. Analysis of the insertion sites at the nucleotide level demonstrated a biased pattern of insertion into regions rich in stretches of A's and T's. Although Tn916 integrated at multiple dispersed positions throughout the chromosome, 9 of 10 insertion events occurred in noncoding, intergenic DNA. It was determined that the intergenic DNA was over 5% more A + T-rich than that of protein coding sequences. This suggests that A + T-rich sequences similar to the Tn916 insertion site would be more likely to reside in the intergenic DNA. In an effort to identify other likely sites for transposon integration, a hidden Markov model of the consensus target insertion site was derived from the Tn916-H. influenzae junction fragments and searched against the entire genome. Eighty percent of the 30 highest-scoring predicted Tn916 target sites were from intergenic, nonprotein-coding regions of the genome. These data support the hypothesis that Tn916 has a marked preference for insertion into noncoding DNA for H. influenzae, suggesting that this mobile element has evolved to minimize disruption of host cell function on integration.

Base Sequence↗
PubMed2004

Predicted values of propofol EC50 and sevoflurane concentration for insertion of laryngeal mask Classic and ProSeal.

BACKGROUND: A new laryngeal mask airway, the ProSeal (PLMA), is said to be more difficult to insert than the laryngeal mask airway Classic (CLMA) using propofol anaesthesia. Therefore, we expected a greater dose of propofol and sevoflurane to be required to insert the PLMA compared with the CLMA. We determined the effective concentration 50% (EC(50)) of propofol and end-tidal sevoflurane to allow insertion of the PLMA and the CLMA. METHODS: Seventy-six elective female patients (aged 20-60 yr and ASA I-II) were randomly assigned to one of four groups. Either a PLMA or a CLMA was inserted using either propofol target controlled infusion or sevoflurane. Both propofol and sevoflurane targets were determined with a modified Dixon's up-and-down method. After equilibration between the predetermined blood and effect site concentrations, which had been held steady for more than 10 min, LMA insertion was attempted without neuromuscular block. RESULTS: The predicted EC(50CLMA) and EC(50PLMA) for propofol were 3.14 (0.33) and 4.32 (0.67) micro g ml(-1). E'(CLMA) and E'(PLMA) of sevoflurane (mean (SD)) were 2.36 (0.22) and 2.82 (0.45)% (P<0.01 and 0.05, respectively). CONCLUSIONS: The estimated concentration of propofol and the sevoflurane concentration needed to allow insertion of the ProSeal are respectively 38 and 20% greater than those needed for insertion of the Classic LMA.

Adult↗
PubMed1997

SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane.

SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.

Adenosine Triphosphatases↗
PubMed2001

Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA.

Oxa1p is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1p has been shown previously to directly facilitate the export of the N-terminal domains of membrane proteins across the inner membrane to the intermembrane space of mitochondria. Here we report on a general role of Oxa1p in the membrane insertion of proteins. (i) The function of Oxa1p is not limited to the insertion of membrane proteins that undergo N-terminal tail export; rather, it also extends to the insertion of other polytopic proteins such as the mitochondrially encoded Cox1p and Cox3p proteins. These are proteins whose N-termini are retained in the mitochondrial matrix. (ii) Oxa1p interacts directly with these substrates prior to completion of their synthesis. (iii) The interaction of Oxa1p with its substrates is particularly strong when nascent polypeptide chains are inserted into the inner membrane, suggesting a direct function of Oxa1p in co-translational insertion from the matrix. Taken together, we conclude that the Oxa1 complex represents a general membrane protein insertion machinery in the inner membrane of mitochondria.

Carrier Proteins↗