Soret-excited Raman spectroscopy of the spinach cytochrome b6f complex. Structures of the b- and c-type hemes, chlorophyll a, and beta-carotene.

Soret-excited resonance Raman (RR) spectra of the spinach cytochrome b6f complex (cyt b6f) are reported for the oxidized, native, ascorbate-reduced, and dithionite-reduced forms. Using excitations at 441.6, 413.1, and 406.7 nm, RR contributions of chlorophyll a, beta-carotene, the c-type heme of cytochrome f, and the b-type hemes of cytochrome b6 of the b6f complex were identified and the data compared to those previously obtained for the Rhodospirillum rubrum bc1 complex [Le Moigne, C., Schoepp, B., Othman, S., Verméglio, A., and Desbois, A. (1999) Biochemistry 38, 1066-1076]. RR bands arising from the b(6)f-associated chlorophyll a and beta-carotene pigments were found to be particularly intense in the spectra excited at 441.6 nm. The frequencies of the phorbin skeleton of chlorophyll a at 1606, 1552, and 1525 cm(-1) are typical of a Mg atom with a single axial ligand. Strong RR bands corresponding to stretching or deformation modes of beta-carotene were detected at 1137, 1157, 1191, 1216, and 1531 cm(-1) in the different forms of cyt b6f. This set of frequencies is assigned to an all-trans configuration of the polyene chain. The redox titrations of the b(6)f complex allow the characterization of RR bands of the three hemes. The nu10, nu2, nu3, and nu8 modes of reduced cyt f are detected at 1619, 1591, 1492, and 356 cm(-1), respectively. From this set of frequencies, one can conclude that the particular histidine/amine heme coordination found in the truncated soluble domain of cyt f is a specific feature of the entire cyt f included in the b6f complex. The frequencies of the nu2, nu8, and nu10 marker modes are consistent with different conformations for the two b-type hemes of cyt b6f. One of these hemes is strongly distorted (nu2, nu8, and nu10 at 1581, 351, and 1610 cm(-1), respectively), while the other one is planar (1586, 345, and 1618 cm(-1), respectively). Largely different structures for the b-type hemes appear to be a common property for the bc1/b6f complexes.

Probing of the membrane topology of sarcoplasmic reticulum Ca2+-ATPase with sequence-specific antibodies. Evidence for plasticity of the c-terminal domain.

The topology of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles was investigated with the aid of sequence-specific antibodies, produced against oligopeptides corresponding to sequences close to the membranous portions of the protein. The antisera in competitive enzyme-linked immunosorbent assays only reacted with intact SR vesicles to a limited extent, but most epitopic regions were exposed by low concentrations of nondenaturing detergent, octaethylene glycol dodecyl ether (C12E8) or after removal of cytosolic regions by proteinase K. In particular, these treatments exposed the loop regions in the C-terminal domain, including L7-8, the loop region located between transmembrane segments M7 and M8, with a putative intravesicular position, which had immunochemical properties very similar to those of the C terminus with a documented cytosolic exposure. In contrast to this, the reactivity of the N-terminal intravesicular loop regions L1-2 and L3-4 was only increased by C12E8 treatment but not by proteinase K proteolysis. Complexation of Ca2+-ATPase with beta,gamma-CrATP stabilized the C-terminal domain of Ca2+-ATPase against proteinase K proteolysis and reaction with most of the antisera, but immunoreactivity was maintained by the L6-7 and L7-8 loops. Immunoelectron microscopic analyses of vesicles following negative staining, thin sectioning, and the SDS-digested freeze-fracture labeling method suggested that the L7-8 epitope, in contrast to L6-7 and the C terminus, can be exposed on either the intravesicular or cytosolic side of the membrane. A preponderant intravesicular location of L7-8 in intact vesicles is suggested by the susceptibility of this region to proteolytic cleavage after disruption of the vesicular barrier with C12E8 and in symmetrically reconstituted Ca2+-ATPase proteoliposomes. In conclusion, our data suggest an adaptable membrane insertion of the C-terminal Ca2+-ATPase domain, which under some conditions permits sliding of M8 through the membrane with cytosolic exposure of L7-8, of possible functional significance in connection with Ca2+ translocation. On the technical side, our data emphasize that extreme caution is needed when using nondenaturing detergents or other treatments like EGTA at alkaline pH to open up vesicles for probing of intravesicular location with antibodies.

Do transmembrane segments in proteolyzed sarcoplasmic reticulum Ca(2+)-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K?

The present study was undertaken to investigate the Ca2+ binding properties of sarcoplasmic reticulum Ca(2+)-ATPase after removal of the cytoplasmic regions by treatment with proteinase K. One of the proteolysis cleavage sites (at the end of M6) was found unexpectedly close to the predicted membrane-water interphase, but otherwise the cleavage pattern was consistent with the presence of 10 transmembrane ATPase segments. C-terminal membranous peptides containing the putative transmembrane segments M7 to M10 accumulated after prolonged proteolysis, as well as large water-soluble fragments containing most of the phosphorylation and ATP-binding domain. Ca2+ binding was intact after cleavage of the polypeptide chain in the N-terminal region, but cuts at other locations disrupted the high affinity binding and sequential dissociation properties characteristic of native sarcoplasmic reticulum, leaving the translocation sites with only weak affinity for Ca2+. High affinity Ca2+ binding could only be maintained when proteolysis and subsequent manipulations took place in the presence of a Ca2+ concentration high enough to ensure permanent occupation of the binding sites with Ca2+. We conclude that in the absence of Ca2+, the complex of membrane-spanning segments in proteolyzed Ca(2+)-ATPase is labile, probably because of relatively free movement or rearrangement of individual segments. Our study, which is discussed in relation to results obtained on Na+,K(+)-ATPase and H+,K(+)-ATPase, emphasizes the importance of the cytosolic segments of the main polypeptide chain in exerting constraints on the intramembranous domain of a P-type ATPase.

Elongation factor 1 alpha (EF-1 alpha) is concentrated in the Balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenopus laevis.

In Xenopus laevis oocytes two distinct systems catalyze the mRNA-dependent binding of aminoacyl tRNA to the A site of ribosomes. These systems are elongation factor 1 alpha (EF-1 alpha) and the 42S nucleoprotein particle. This particle is also implicated in the long-term storage of 5S RNA and aminoacyl tRNA during early oogenesis. We report here that the ribosomes and the storage particles are distributed uniformly in the cytoplasm of previtellogenic (stage I) oocytes. In contrast, EF-1 alpha is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body. When the Balbiani body disperses in early vitellogenic oocytes (stage II), EF-1 alpha becomes evenly distributed in the cytoplasm. The main phase of EF-1 alpha accumulation follows the disappearance of the 42S particles (stage II), but coincides with the main phase of ribosome accumulation (stages III and IV).