Reconstitution of protein translocation activity from partially solubilized microsomal vesicles

We have used a reconstitution assay to demonstrate that protein translocation activity can be recovered after microsomal vesicles derived from the rough endoplasmic reticulum have been partially solubilized with n-octyl-beta-glucopyranoside. Two independent approaches were used to establish conditions for partially solubilizing microsomal membranes. When the lipid bilayer was disrupted by detergents to the extent that the integrity of the lipid bilayer had been perturbed, membranes were inactive for translocation. However, detergent-treated membranes could be reconstituted in good yield into a translocation competent form once the detergent was removed.     

DCCD inhibits protein translocation into plasma membrane vesicles from Escherichia coli at two different steps.

In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). DCCD was inhibitory to both co- and post-translational translocations, suggesting an involvement of the H+-translocating F1F0-ATPase in either mode of transport. This was verified by (i) the dependence of efficient co-translational translocation upon a low salt, i.e. F1-containing extract from membrane vesicles; (ii) the co-purification of the translocation activity present in this extract and F1-ATPase; (iii) the inability of either vesicles or their low-salt extract, derived from F1F0-ATPase-lacking mutant strains, to support translocation; and (iv) the greatly diminished extent of ATP-dependent, post-translational translocation into F1-deprived vesicles. Membranes devoid of F1 did show, however, residual translocation activity that was also found to be inhibitable by DCCD. These results suggest a dual target for DCCD in bacterial protein export, one being the H+-ATPase and the other an as yet unidentified translocation factor.     

Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process

The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.     

Identification and purification of a protein that binds the yeast ARS consensus sequence

We have identified a yeast protein that binds specifically to the ARS consensus sequence. By two-step chromatography we have purified the factor to apparent homogeneity as a single polypeptide of 67 kd. The purified ARS consensus-binding protein (ACBP) recognizes the ARS consensus of the four genomic ARS elements tested, binding preferentially to the T-rich single strand. Point mutations in the consensus significantly reduce the affinity of the single-strand binding. At the histone H4 ARS, ACBP recognizes both the perfect ARS consensus and a 9/11 match 3' of it. These two binding sites correlate with the boundaries of the minimal functional H4 ARS element. A similar configuration of binding sites is found at ARS1. We propose a model implicating this factor in an early step of the initiation of DNA replication.     

SecA protein is required for secretory protein translocation into E. coli membrane vesicles

The soluble and membrane components of an E. coli in vitro protein translocation system prepared from a secA amber mutant, secA13[Am], contain reduced levels of SecA and are markedly defective in both the cotranslational and posttranslational translocation of OmpA and alkaline phosphatase into membrane vesicles. Moreover, the removal of SecA from soluble components prepared from a wild-type strain by passage through an anti-SecA antibody column similarly abolishes protein translocation. Translocation activity is completely restored by addition of submicrogram amounts of purified SecA protein, implying that the observed defects are solely related to loss of SecA function. Interestingly, the translocation defect can be overcome by reconstitution of SecA into SecA-depleted membranes, suggesting that SecA is an essential, membrane-associated translocation factor.     

Effects of antibiotics and other inhibitors on ATP-dependent protein translocation into membrane vesicles.

The effects of several membrane antibiotics and other agents on ATP-dependent protein translocation were examined in membrane vesicles under conditions where no significant proton motive force was present. The membrane perturbants ethanol and procaine abolished ATP-dependent protein translocation. Phenethyl alcohol at low concentrations abolished translocation, whereas at high concentrations it allowed precursors to be translocated but inhibited their processing. Translocation of precursors promoted by phenethyl alcohol was temperature dependent and occurred without an added energy source but was enhanced by ATP. However, such precursors could not be further processed to mature forms upon removal of the alcohol. The membrane-active antibiotics polymyxin B and gramicidin S were strong inhibitors of translocation, whereas gramicidin D, cerulenin, and mycobacillin had no effect even at higher concentrations, indicating some specificity in interference with protein translocation. Duramycin, an antibiotic previously shown to affect protein-lipid interaction, severely impaired protein translocation. These results showed that membrane structures play important roles, either directly or indirectly, in protein translocation. Chelating agents 1,10-phenanthroline and EDTA, but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], also abolished protein translocation.     

Bipartite signal sequence mediates nuclear translocation of the plant potyviral NIa protein.

The NIa protein of certain plant potyviruses localizes to the nucleus of infected cells. Previous studies have shown that linkage of NIa to reporter protein beta-glucuronidase (GUS) is sufficient to direct GUS to the nucleus in transfected protoplasts and in cells of transgenic plants. In this study, we mapped sequences in NIa that confer karyophilic properties. A quantitative transport assay using transfected protoplasts, as well as in situ localization technique using epidermal cells from transgenic plants, were employed. Two domains within NIa, one between amino acid residues 1 to 11 (signal domain I) and the other between residues 43 to 72 (signal domain II), were found to function additively for efficient localization of fusion proteins to the nucleus, although either region independently could facilitate a low level of translocation. Like signals from animal cells, both nuclear transport domains of NIa contain a high concentration of basic (arginine and lysine) residues. Nuclear transport signal domain II overlaps or is very near Tyr62, which is the residue that mediates covalent attachment of a subset of NIa molecules to the 5' terminus of viral RNA within infected cells. The nature of the NIa nuclear transport signal and the possibility for regulation of NIa translocation are discussed.     

Sordarin inhibits fungal protein synthesis by blocking translocation differently to fusidic acid.

Sordarin derivatives are selective inhibitors of fungal protein synthesis, which specifically impair elongation factor 2 (EF-2) function. We have studied the effect of sordarin on the ribosome-dependent GTPase activity of EF-2 from Candida albicans in the absence of any other component of the translation system. The effect of sordarin turned out to be dependent both on the ratio of ribosomes to EF-2 and on the nature of the ribosomes. When the amount of EF-2 exceeded that of ribosomes sordarin inhibited the GTPase activity following an inverted bell-shaped dose-response curve, whereas when EF-2 and ribosomes were in equimolar concentrations sordarin yielded a typical sigmoidal dose-dependent inhibition. However, when ricin-treated ribosomes were used, sordarin stimulated the hydrolysis of GTP. These results were compared with those obtained with fusidic acid, showing that both drugs act in a different manner. All these data are consistent with sordarin blocking the elongation cycle at the initial steps of translocation, prior to GTP hydrolysis. In agreement with this conclusion, sordarin prevented the formation of peptidyl-[(3)H]puromycin on polysomes from Candida albicans.     

Suppressor mutations that restore export of a protein with a defective signal sequence

A selection procedure is described that should allow the genetic identification of cellular components involved in the process of protein localization in Escherichia coli. This procedure makes use of mutations that alter the signal sequence of the λ receptor protein (product of the lamB gene), and prevent export of this protein to its normal outer membrane location. Several suppressor mutations have been identified that restore export of the mutant λ receptor protein. Mapping experiments show that the suppressor phenotype is the result of mutations in any of at least three different chromosomal loci. One class of suppressor mutations, the class containing the largest number of independent isolates, maps in the major ribosomal gene cluster, suggesting that the suppressor phenotype is the consequence of an altered ribosomal protein. This class of suppressors phenotypically suppresses all known export-defective mutations, internal to the signal sequence region of the lamB gene. These results suggest that ribosomes play an important role in the export of λ receptor to the outer membrane.     

The motif of SPARC that inhibits DNA synthesis is not a nuclear localization signal

SPARC (secreted protein acidic and rich in cysteine), although primarily known as a secreted, matricellular protein, has also been identified in urothelial cell nuclei. Many biological activities, including inhibition of cell adhesion and repression of DNA synthesis, have been ascribed to SPARC, but the influence of its intracellular localization on each of these activities is unknown. When exposed by epitope retrieval and nuclear matrix unmasking techniques, endogenous SPARC was found to localize strongly to the nuclei and the nuclear matrix of cultured urothelial cells. Live-cell time-lapse imaging revealed that exogenous fluorescently labeled recombinant (r) SPARC was taken up from medium over a 16 h period and accumulated inside cells. Two variants of rSPARC with alterations in its putative nuclear localization signal (NLS) were generated to investigate the existence and effects of the NLS. These variants demonstrated similar biophysical characteristics as the wild-type protein. Visualization by a variety of techniques, including live-cell imaging, deconvolution microscopy, and cell fractionation, all concurred that exogenous rSPARC was not able to localize to cell nuclei, but instead accumulated as perinuclear clusters. Localization of the rSPARC NLS variants was no different than wild-type, arguing against the presence of an active NLS in rSPARC. Imaging experiments showed that only permeabilized, dead cells avidly took up rSPARC into their nuclei. The rSPARC(no NLS) variant proved ineffective at inhibiting DNA synthesis, whereas the rSPARC(strong NLS) variant was a more potent inhibitor of DNA synthesis than was wild-type rSPARC. The motif of SPARC that inhibits the synthesis of urothelial cell DNA is therefore not a nuclear localization signal, but its manipulation holds therapeutic potential to generate a "Super-SPARC" that can quiesce proliferative tissues.     

Segregation of the signal sequence receptor protein in the rough endoplasmic reticulum membrane

The signal sequence receptor (SSR), an integral membrane glycoprotein of 34 kDa, has previously been shown to be a component of the molecular environment which nascent polypeptide chains meet in passage through the endoplasmic reticulum (ER) membrane. We have used antibodies directed against the SSR and both immunocytochemistry and cell fractionation to determine its distribution in rat liver cells. SSR was found largely restricted to the rough ER. Only small amounts of the protein were detected in smooth ER. These results provide further evidence for a functional differentiation of rough and smooth ER and for a role of SSR in protein translocation across the ER membrane.     

Monte Carlo simulations of voltage-driven translocation of a signal sequence

CD1d-deficient (CD1d-/-) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a V(alpha)19.1-J(alpha)26 (AV19-AJ33) invariant TCR alpha chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1+ but not NK1.1- T cell population isolated from CD1d-/- mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d-/- NKT cells expressed the V(alpha)19.1-J(alpha)26 invariant TCR alpha chain. The expression of the invariant V(alpha)19.1-J(alpha)26 mRNA was absent in beta2-microglobulin-deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR alpha chain (V(alpha)19.1-J(alpha)26) that is selected by an MHC class I-like molecule other than CD1d.     

Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energized membrane and not simply from ATP. Treatment of the vesicles with protease, under conditions that did not interfere with subsequent protein synthesis, also inactivated them for subsequent translocation. We conclude that export of some proteins requires protein-containing machinery in the cytoplasmic membrane that derives energy from the proton motive force.     

Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles.

The involvement of possible cytoplasmic factors in ATP-dependent postttranslational translocation of proteins into Escherichia coli membrane vesicles was examined. The precursor of OmpA protein was partially purified by DEAE-cellulose chromatography, and its translocation was found to require material from the soluble cytoplasmic fraction. The fractionated active cytoplasmic translocation factor (CTF) was protease sensitive, micrococcal nuclease insensitive, N-ethylmaleimide resistant, and heat labile. The heat sensitivity of the CTF allowed its specific and preferential inactivation in the crude-precursor synthesis mixture, which provided a simple and rapid assay procedure for the factor during purification. Two active fractions were detected upon further fractionation: the major one was about 8S in sucrose gradient centrifugation and 120 kilodaltons by Sephadex filtration, whereas the other was about 4S and 60 kilodaltons in sucrose gradient centrifugation and by Sephadex filtration, respectively. The active fractions could also be fractionated by DEAE-Sepharose chromatography. These CTFs are apparently different from the previously reported 12S export factor (M. Muller and G. Blobel, Proc. Natl. Acad. Sci. USA 81:7737-7741, 1984).     

Effect of transforming RNA on the synthesis of a protein with a secretory signal sequence in vitro.

U5 small nuclear RNA itself can act as a clastogenic and transforming agent when transfected into cells. In the previous work, the 3' half of the U5 small nuclear RNA first stem structure (designated RNA3S) was capable of driving normal cells into tumorigenic cells when expressed with a poly(A) tail (RNA3S+). This transformation critically depended upon the polypurine sequence GGAGAGGAA in RNA3S+. In this work, we first examined the pre-beta-lactamase and luciferase (model secretory and nonsecretory proteins) translation with the in vitro synthesized RNA3S in rabbit reticulocyte lysate. The capped RNA3S with a poly(A) tail suppressed the translation. In addition, the polypurine sequence played a crucial role in affecting the secretory protein synthesis, indicating a primary action of RNA3S+. Further studies revealed that the oligodeoxynucleotides, corresponding to the polypurine and its antisense sequences, directly contacted 28 S rRNA in ribosome and 7SL RNA in signal recognition particle, respectively, and differentially affected the nascent chain elongation of secretory protein synthesis. These results suggest that RNA3S+ blocks a physiological regulatory function played by signal recognition particle and the ribosome in the secretory protein synthesis and support the idea that the transformation might result from a repressed cellular activity.     

Delta psi stimulates membrane translocation of the C-terminal part of a signal sequence.

For several proteins in Escherichia coli it has been shown that the protonmotive force (pmf) dependence of translocation can be varied with the signal sequence composition, suggesting an effect of the pmf on the signal sequence. To test this possibility, we analyzed the effect of the membrane potential on translocation of the signal sequence. For this purpose, a precursor peptide was used (SP+7), corresponding to the signal sequence of PhoE with the first seven amino acids of the mature part that can be processed by purified leader peptidase. Translocation was studied in pure lipid vesicles containing leader peptidase, with its active site inside the vesicles. In the presence of a positive inside Delta psi, the amount of processing of SP+7 was significantly higher than without a Delta psi, indicating that the translocation of the cleavage region is stimulated by Delta psi. Replacement of the helix-breaking glycine residue at position -10 in the signal sequence for a leucine abolished the effect of Delta psi on the translocation of the cleavage region. It is concluded that Delta psi directly acts on the wild type signal sequence by stimulating the translocation of its C terminus. We propose that Delta psi acts on the signal sequence by stretching it into a transmembrane orientation.