CCCP-treated Cells Research Articles

Co-translational excision of alpha-glucose and alpha-mannose in nascent vesicular stomatitis virus G protein.

Membrane bound polysomes were prepared from HeLa cells infected with vesicular stomatitis virus (VSV), after pulse labeling with [3H]mannose for various times from 15 to 90 min. Oligosaccharides on nascent chains were released from peptides by treatment with endoglycosidase H and sized by high resolution Biogel P4 chromatography. Processing on some nascent chains proceeded to the removal of all three types of alpha-linked glucose and one alpha-1,2-mannose from the Glc3Man9GlcNAc precursor showing that the enzymes responsible were not only active on nascent chains but were present in the rough endoplasmic reticulum (RER). Incubation of cells for various times in cycloheximide, where chain elongation had ceased, made no difference to the profile of oligosaccharides on the nascent chains, and trimming proceeded no further than Man8GlcNAc2Asn . Carbonyl cyanide m-chlorophenylhydrazone (CCCP), an energy inhibitor reportedly able to block the transfer of glycoproteins from the RER, increases the amount of Man8-oligosaccharides on the nascent chains and also the amount of Glc3Man9GlcNAc precursor. On completed G protein in the RER fraction from which membrane bound polysomes were prepared, processing occurred to Man6 - but not to Man5GlcNAc sized oligosaccharides in the CCCP-treated cells. By contrast, processing to Man5GlcNAc oligosaccharides was observed in unfractionated control cells.

Biosynthesis and topogenesis of aspartate aminotransferase isoenzymes in chicken embryo fibroblasts. The precursor of the mitochondrial isoenzyme is either imported into mitochondria or degraded in the cytosol.

In chicken embryo fibroblasts pulsed wih [35S]methionine, a precursor of mitochondrial aspartate aminotransferase with higher molecular weight (delta Mr approximately 3000) was detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping of the precursor and the mature enzyme confirmed their precursor-product relationship. No precursor of the homologous cytosolic isoenzyme was found. The precursor of the mitochondrial isoenzyme is synthesized on membrane-free polysomes in the cytosol (Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345); its half-life is 30 to 60 s. The pronounced susceptibility of the precursor toward exogenous proteases contrasts the stability of the mature enzyme and thus indicates that the conformation or the quarternary structure of the protein must change concomitantly with its import into mitochondria. Administration of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to the cell cultures blocks the import of many matrix and inner membrane proteins into mitochondria. The precursor of mitochondrial aspartate aminotransferase is found to be accumulated in the cytosol. However, its steady state concentration in CCCP-treated cells exceeds the concentration in untreated cells by not more than 1 order of magnitude. During a chase, the radioactive precursor disappears with a half-life of approximately 5 with min without formation of mature enzyme. Thus, in CCCP-treated cells, a degradative process is limiting the accumulation of the precursor in the cytosol. When the chase is performed in the presence of cysteamine, an antagonist of CCCP, the precursor is processed to the mature enzyme. Newly synthesized cytosolic aspartate aminotransferase is not degraded.

Initiation of flagella rotation in Rhodopseudomonas sphaeroides

Flagellate bacteria move by rotating semi-rigid helical flagella in either a clockwise or anticlockwise direction, using the frequency of switching between the two directions of rotation to bias their overall movement in a favourable direction (Macnab, 1978). It is probable that flagellar rotation involves the movement of protons down the membrane proton gradient, between two complex protein rings in the cytoplasmic membrane. One protein ring appears to be fixed to the flagellum and free to rotate, and the other is fixed in the membrane. The movement of protons via an unidentified proton pore in the ring system causes the two rings to move against each other. This rotation can use either the electrical or chemical potential to power the rotation (Khan & Macnab, 1980). Although the energetics of flagellar rotation have been investigated, the mechanism by which flagellar rotation is initiated, or the rotation in one direction is stopped and restarted in the opposite direction, by using a unidirectional proton gradient, is unknown. Photosynthetic bacteria become non-motile if incubated anaerobically in the dark, but regain motility upon actinic illumination. If a useful protonmotive force is the only requirement for initiation of flagellar rotation, as is assumed for its continued rotation, then the time taken for motility to resume upon illumination after incubation in the dark should be similar to the time for the creation of a stable increased membrane potential. Rhodopseudomonas sphaeroides possesses an endogenous indicator of membrane potential, the carotenoids. When there is a change in the potential across the cytoplasmic membrane, there is an electrochromic shift in the absorbance of the carotenoids (Jackson & Crofts, 197 1). Photosynthetically grown R . sphaeroides were isolated into either Hepes [4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid1 or potassium phosphate buffer (lmM, pH7.0) with IOpM-disodium EDTA. After several hours anaerobic incubation in the dark, the bacteria were actinically illuminated and the carotenoid bandshift was measured in an Aminco dual-wavelength spectrophotometer as previously described (Armitage & Evans, 1979). There was a very fast rise in membrane potential as measured by the carotenoid bandshift (half-time, ti 5 5 ms). Similarly, if the bacteria were incubated in 1 O p ~ buffer for several hours in the dark in an anaerobic stirred vessel there was a decrease of about 0.1 pH unit within 15 s of illumination. However, when R. sphaeroides, isolated under similar conditions and sealed into micro-slides (50mm, with O.lmm pathlength), was examined with similar illumination conditions on the microscope, the first bacterium did not start to move for several seconds and the normal percentage of motile bacteria was not observed until more than 60s illumination. The effect could be enhanced by the addition of low concentrations of uncouplers. If 1 ~ M C C C P ' (final concn.) was added to the bacteria in the dual-wavelength spectrophotometer, there was a gradual blue shift in carotenoid absorbance taking several minutes. When the new baseline absorbance stabilized (the decrease in potential being presumably the result of residual respiration in the dark), the actinic light-induced bandshift remained unaltered or increased. The bacteria therefore retained their ability to maintain a light-induced membrane potential. Measurement of external pH under similar conditions showed the same result as for untreated bacteria. However, examination microscopically of the CCCP-treated cells showed an increase in the time taken for motility to resume. It was now several minutes before the first bacterium moved, and many more before 50% were motile. When motility restarted, individual bacteria moved at normal speed and their behaviour was similar to that of untreated bacteria. The length of the delay was related to the concentration of uncoupler, which could also be related to the decrease in the size of the bandshift as the concentration of uncoupler was increased. As the light-induced membrane potential, measured by the bandshift, decreased, the time taken for motility to resume increased (16 s for first motile bacterium at 0.5 ~ M C C C P , 30s at l p ~ , 65s at 5pM). However, the time taken for motility to resume at each uncoupler concentration was independent of the length of dark incubation after uncoupler addition, the maximum delay remaining constant after 12 min dark incubation. The length of the delay was also pH-dependent; at pH6.5 O. I~M-CCCP caused a delay of 2min, whereas at pH7.5 the same concentration only delayed the onset of motility by 7 s. These results suggest that the protonmotive force alone is not enough for the initiation of flagellar rotation, but there is an additional potential-dependent component, possibly a protein modification. It is possible that this is also required for the switching mechanism, as bacteria with artificially low membrane potentials have been shown to be unable to change direction (Khan & Macnab, 1980).

Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli.

The leucine-specific binding protein of Escherichia coli is a periplasmic protein that is synthesized as a precursor and subsequently is processed during its secretion into the periplasmic space. The processing of both the leucine-specific binding protein and a plasmid-coded beta-lactamase is inhibited by phenethyl alcohol and by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP). The levels of CCCP that inhibit processing also produce significant decreases in the membrane potential. Valinomycin, a potassium ionophore, also inhibits processing of the leucine-specific binding protein in spheroplasts. Processing can be restored in CCCP-treated cells and in valinomycin-treated spheroplasts by dilution of the treated cells in fresh medium. These results suggest a role for membrane potential in the secretion of periplasmic proteins. A model is presented which suggests that membrane potential plays a primary role in the proper orientation of the precursor signal sequence within the membrane, thus promoting processing and secretion.