Amino acid identities in the three redox center-carrying polypeptides of cytochromebc 1/b 6 f complexes

The comparison of primary structures is extended to 22 cytochromesb orb ₆, 12 cytochromesc ₁ orf, and 8 Rieske FeS proteins. Conclusions are drawn as to their phylogenetic relationship as well as on conserved, functionally important amino acids and secondary structures. The results are in favor of two independent quinone binding sites at opposite surfaces of the membrane, topping one of the two hemes of cytochromeb each.     

The major light-harvesting complex of Photosystem II: aspects of its molecular and cell biology

The light-harvesting complex of photosystem II (LHC II) contains one major (LHC IIb) and at least three minor chlorophyll-protein components. The apoproteins of LHC IIb (LHCP) are encoded by nuclear genes and synthesized in the cytoplasm as a higher molecular weight precursor(s) (pLHCP). Several genes coding for pLHCP have been cloned from various higher plant species. The expression of these genes is dependent upon a variety of factors such as light, the developmental stage of the plastids and the plant. After its synthesis in the cytoplasm, pLHCP is imported into plastids, inserted into thylakoids, processed to its mature form, and assembled into LHC IIb. The pathway of assembly of LHC IIb in the thylakoid membranes is currently being investigated in several laboratories. We present a model that gives some details of the steps in the assembly process. Many of the steps involved in the synthesis and assembly are dependent on light and the stage of plastid development.Abbreviations PS Photosystem - LHC II Light-harvesting complex of PS II - LHCP Apoproteins of LHC IIb - pLHCP Precursor of LHCP - PAGE Polyacrylamide gel electrophoresis     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

Analysis of nuclear proteins from silk glands ofBombyx mori

A gentle method for the isolation of nuclei from developing silk glands ofBombyx mori has been standardized. The nuclei, whether isolated or directly visualizedin situ within the silk glands, exhibit complex morphology. The nuclei occupy almost the entire volume of the gigantic silk gland cells. Although the isolated nuclei still retain their ramified morphology, being polyploid they are fragile and often become fragmented. The histone and low-salt-extractable proteins from nuclei isolated from the middle and posterior silk glands on different days of the fourth and fifth instars of larval development have been analysed. The histones did not show any stage- or tissue-specific variations whereas the low-salt-extractable proteins showed some developmental stage specific variation. Using the antibody raised against one such protein, its absence in the early stage of development has been confirmed by Western blotting techniques. This developmental stage specific protein may be functionally linked to some activities responsible for boosting up the production of silk or silk-related proteins during the fifth instar of larval development.     

Structural similarities among the high molecular weight protein fractions of oilseeds

Data on the physico-chemical properties of proteins from soybean, groundnut, sesame seed, sunflower seed, safflower seed, mustard seed, rapeseed and cotton seed are fairly extensive. An examination of the available data on high molecular weight proteins suggests that there are similarities in many of their properties. In this report the similarity in amino acid composition, size and shape, molecular weight, secondary structure, subunit composition, association-dissociation at high and low pH, stability towards denaturants, hydrolysis by enzymes and quaternary structure of the high molecular weight proteins is discussed. Based on these similarities a model has been proposed for the associationdissociation, denaturation and reassociation behaviour of the high molecular weight proteins of oilseeds.     

Levels and Distribution of the Calcium-Modulated Proteins S100 and Calmodulin in Rat C6 Glioma Cells

To understand better the mechanisms involved in the transduction of a calcium signal into an intracellular response via multiple calcium-modulated proteins, we have examined the calcium-modulated proteins, S100 and calmodulin, and their intracellular targets in rat C6 glioma cells. Subconfluent, confluent, and postconfluent C6 cells contain predominantly, if not exclusively, the S100 beta polypeptide. The level of S100 beta in C6 cells increases approximately 20-fold from subconfluency to postconfluency whereas the level of calmodulin increases only about two-fold. The subcellular distribution of S100 beta and calmodulin in mitotic cells is similar. However, the subcellular distribution of these proteins in interphase cells is different and appears to change with cell density. Gel overlay analysis demonstrated that the S100- and calmodulin-binding protein profiles are significantly different and that some of the binding proteins appear to change in intensity with cell density. These data demonstrate that S100 beta is the predominant S100 polypeptide in C6 cells and suggest that changes in S100 beta and S100 beta-binding proteins may be involved in regulating S100-mediated intracellular processes in C6 cells. Our studies also suggest that the levels of S100 and calmodulin may be differentially regulated in C6 cells.     

Tubulin isotypes and their interaction with microtubule associated proteins

Summary To assay the functional significance of the multiple but closely related - and -tubulin polypeptides (termed isotypes) that are expressed in mammalian cells, we have generated a number of sera that uniquely discriminate among these isotypes. These sera have been used to demonstrate that there is no subcellular sorting of either - or -tubulin isotypes among microtubules of diverse function, either in cells growing in culture or in tissues consisting of cell types that contain specialized kinds of microtubule. In spite of this failure to segregate between functionally distinct kinds of microtubule, the fact that isotype-specific amino acid sequences have been strictly conserved over extensive periods of evolutionary time argues persuasively for a functional role for the different tubulin gene products. One possibility is that they are required for specific interactions with microtubule associated proteins (MAPs), and that tubulin isotypes have coevolved with different cell type-specific MAPs with which they must interact. We have tested this hypothesis by examining the distribution of -tubulin isotypes in mammalian cerebellum in relationship to the known patterns of expression of a number of MAPs, and find that these patterns correlate in the case of M 2 and MAP 3, and M 6 and MAP 1 a. These data, plus emerging data based on a structural analysis of tau, MAP 1 b and MAP 2 obtained via sequence determination of cloned cDNAs, are discussed in terms of the possible functional significance of tubulin isotype/MAP interactionsin vivo.