Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration

The progression of several human neurodegenerative diseases is characterized by the appearance of intracellular inclusions or cytoskeletal abnormalities. An important question is whether these abnormalities actually contribute to the degenerative process or whether they are merely manifestations of cells that are already destined for degeneration. We have conducted a large screen in Drosophila for mutations that alter the growth or differentiation of cells during eye development. We have used mitotic recombination to generate patches of homozygous mutant cells. In our entire screen, mutations in only two different loci, burned (bnd) and scorched (scrd), resulted in eyes in which the mutant patches appeared black and the mutant tissue appeared to have undergone degeneration. In larval imaginal discs, growth and cell fate specification occur normally in mutant cells, but there is an accumulation of F-actin. Mutant cells degenerate much later during the pupal phase of development. burned mutations are allelic to mutations in the previously described cpb locus that encodes the beta-subunit of the F-actin capping protein, while scorched mutations disrupt the gene encoding its alpha-subunit (cpa). The alpha/beta-heterodimer caps the barbed ends of an actin filament and restricts its growth. In its absence, cells progressively accumulate actin filaments and eventually die. A possible role for their human orthologs in neurodegenerative disease merits further investigation.     

Beta-subunit of bovine rod photoreceptor cGMP phosphodiesterase. Comparison with the phosphodiesterase family

A group of cDNA clones encoding the beta-subunit of bovine rod photoreceptor cGMP phosphodiesterase were isolated for structural analysis. The encoded polypeptide has 853 residues with a calculated molecular mass of 98 kDa. The beta-subunit is 72% identical to the rod cGMP phosphodiesterase alpha-subunit. Like the alpha-subunit and the cone alpha'-subunit, the beta-subunit belongs to the family of phosphodiesterase genes. The beta- and alpha-subunits are more similar to each other than either is to the cone alpha'-subunit, suggesting either that the beta- and alpha-subunits diverged more recently or that their divergence was restrained by the rod functional environment.     

Three distinct classes of the alpha-subunit of the nuclear pore-targeting complex (importin-alpha) are differentially expressed in adult mouse tissues.

The process of active nuclear protein transport is mediated by the nuclear localization signal (NLS). An NLS-containing karyophile forms a stable complex, termed the nuclear pore-targeting complex, to target nuclear pores. The alpha-subunit of the complex (importin-alpha) binds to the NLS and the beta-subunit (importin-beta) carries the alpha-subunit, bound to the NLS substrate, into the nucleus. To date, five mouse alpha-subunits have been identified and classified into three subfamilies (alpha-P, alpha-Q, and alpha-S). The expression of these alpha-subunits and the beta-subunit in various adult mouse tissues was examined by immunoblotting and immunohistochemistry using antibodies specific for each subfamily of the alpha-subunit or the beta-subunit. The beta-subunit was found to be ubiquitously expressed, whereas each subfamily of the alpha-subunit showed a unique expression pattern in various tissues, especially in brain and testis. In brain, the expression of alpha-P was not observed, whereas alpha-S was significantly expressed in Purkinje cells, and pyramidal cells of the hippocampus and cerebral cortex. In testis, alpha-P was expressed predominantly in primary spermatocytes, whereas alpha-Q was found mainly in Leydig cells. Expression of alpha-S was detected in almost all cells in convoluted seminiferous tubules and Leydig cells to a similar extent. These results suggest that nuclear protein import may be controlled in a tissue-specific manner by alpha-subunit family proteins.     

Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.     

Primary structures of the genes, faoA and faoB, from Pseudomonas fragi B-0771 which encode the two subunits of the HDT multienzyme complex involved in fatty acid beta-oxidation.

Three enzyme activities involved in fatty acid beta-oxidation, i.e., those of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, are exhibited by one multienzyme complex (HDT) composed of two molecules each of two peptides in Pseudomonas fragi. Using specific antisera against the two subunits of HDT, we isolated the genes encoding the subunits of HDT and designated them "faoA" (for the alpha-subunit) and "faoB" (for the beta-subunit). Their complete nucleotide sequences were determined and it was revealed that faoA and faoB, both with individual putative S.D. sequences at suitable positions, formed a cluster, in that order. The amino acid sequences deduced from the nucleotide sequences of the two genes indicated that the alpha-subunit, encoded by faoA, is a polypeptide of 715 amino acid residues, and that the beta-subunit, encoded by faoB, consists of 390 amino acid residues lacking the first methionine of the primary product encoded by faoB. Immunoblotting of cell lysates prepared from Escherichia coli transformants carrying plasmids which possess the faoA and/or faoB gene with antisera against the subunits of HDT showed that both the faoA and faoB genes were transcribed and translated in E. coli. The overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase were increased in the E. coli cells transformed with the plasmid possessing the faoA gene, suggesting that both the hydratase and dehydrogenase activities may be exhibited by the alpha-subunit of HDT.(ABSTRACT TRUNCATED AT 250 WORDS)     

西方蜜蜂Lozenge基因的克隆鉴定及时空表达分析

Lozenge蛋白（Lz蛋白）是昆虫的重要转录因子,在昆虫胚胎发育过程中发挥重要作用。为研究Lozenge在西方蜜蜂Apis mellifera中的作用,本研究克隆了Lozenge基因,并对其进行生物信息学分析,同时基于荧光定量PCR技术检测该基因在西方蜜蜂不同发育时期（卵期、幼虫期、蛹期和成年蜂）和10日龄哺育蜂各组织的表达谱。生物信息学分析结果显示,Lozenge基因的开放阅读框（ORF）为1 554 bp,共编码517个氨基酸,预测分子量为54.63918 kDa,等电点为6.08;结构域预测分析发现Lozenge蛋白含有一个Runt结构域,多物种蛋白序列对比发现该蛋白同源性高。时期表达谱表明,该基因在第1日卵和第2日卵的表达量远高于其他时期,在卵期表达量随时间依次递减,幼虫期表达量极低,蛹期表达量呈先增后减的趋势,而成年蜂中均有表达;组织表达谱显示,该基因在哺育蜂头部、上颚腺中的表达量较高,而在腹部的表达量低。这些结果表明,Lozenge基因可能在西方蜜蜂胚胎期细胞发育过程、哺育蜂蜂王浆合成和分泌过程中发挥重要作用,这些结果为该基因功能的深入研究提供了重要的理论参考。     

The primary structure of the beta-subunit of the cell surface adhesion glycoproteins LFA-1, CR3 and p150,95 and its relationship to the fibronectin receptor.

The lymphocyte-function-associated antigen-1 (LFA-1), the complement receptor type 3 (CR3) and the antigen p150,95 are cell-surface glycoproteins. They are heterodimeric complexes, each containing a unique alpha-subunit noncovalently associated with a common beta-subunit. We have purified the beta-subunit from human spleen and obtained limited peptide sequences. What appears to be the complete primary structure for the fully processed beta-subunit was obtained by cDNA sequencing of clones from a phorbol ester (PMA) stimulated U937 cDNA library. There are five possible glycosylation sites and a transmembrane segment. The sequence contains a high level of cysteine (7.6%), with 24 of the 57 cysteine residues being found in three repeating units each with eight residues. The entire primary structure has 47% identity to a subunit of a fibronectin binding protein from chicken fibroblasts. It seems that LFA-1, CR3 and p150,95 antigens may belong to an extended family of cell surface molecules including the fibronectin binding protein.     

Down-regulation of the alpha- and beta-subunits of the calcium-activated potassium channel in human myometrium with parturition

Large-conductance, calcium-dependent potassium (BKCa) channels are implicated in maintaining uterine quiescence during pregnancy. The mechanisms whereby calcium sensitivity of the BKCa channel is dramatically removed at parturition remain unknown. The aim of the present study was to investigate whether this loss of calcium sensitivity of the BKCa channel with the onset of labor is associated with changes in the protein expression of the alpha- and/or beta-subunit or arises from a physical dissociation of the alpha-subunit from the beta-subunit. The beta-subunit is a key determinant of BKCa-channel Ca2+ sensitivity. Western blot analysis, using alpha- and beta-subunit-specific antibodies, detected bands of 110-125 and 36 kDa, respectively. Protein expression levels of the alpha-subunit in term labor myometrium were significantly reduced compared with term pregnancy without labor. Furthermore, alpha-subunit levels at term pregnancy were significantly increased relative to the nonpregnant state, whereas levels at preterm gestations were unchanged. Densitometric analysis demonstrated significantly decreased beta-subunit levels in term and preterm labor samples compared with term nonlabor samples. Immunoprecipitation studies revealed the presence of both the alpha- and beta-subunits in samples taken before or after the onset of labor. We conclude that during labor, the alpha-subunit is not physically uncoupled from the beta-subunit, but a decline occurs in the level of beta-subunit protein, which may underlie the loss of calcium and voltage sensitivity of the BKCa channel with labor. Furthermore, reduced beta-subunit protein in preterm labor myometrium implies that ion channels may also contribute to pathophysiological labor.     

ATP synthase from bovine mitochondria: complementary DNA sequence of the import precursor of a heart isoform of the alpha subunit

The alpha-subunit of ATP synthase from mitochondria is a major component of the extrinsic membrane sector of the enzyme. It is encoded in nuclear DNA. A family of overlapping complementary DNA clones encoding its precursor has been isolated from a bovine library by using in the first instance a mixture of 128 synthetic oligonucleotides designed on the basis of the known protein sequence, and the sequence of the full-length cDNA has been determined. The deduced protein sequence shows that the alpha-subunit of ATP synthase has a presequence of 43 amino acids that is not present in the mature protein. Presumably it directs the protein into the mitochondrial matrix and is removed during the import process. The encoded protein sequence is also longer by one amino acid at its C-terminal end than the protein isolated from F1-ATPase, but this alanine residue may have been removed artifactually during release of the F1-ATPase particle from the inner mitochondrial membrane. With the exception of one uncertainty caused by an ambiguity at one position in the nucleotide sequence, the mature protein sequence encoded in the cDNA is exactly the same as the sequence determined previously by direct analysis of the protein isolated from bovine heart mitochondria [Walker et al. (1985) J. Mol. Biol. 184, 677-701]. The cDNA sequence differs in 158 nucleotides over a region of alignment of 1097 nucleotides from a partial cDNA for the alpha-subunit that has been isolated from a bovine cDNA derived from liver RNA [Breen (1988) Biochem. Biophys. Res. Commun. 152, 264-269].(ABSTRACT TRUNCATED AT 250 WORDS)     

The carboxyl-terminal 161 amino acids of the Na,K-ATPase alpha-subunit are sufficient for assembly with the beta-subunit.

Chimeric cDNAs encoding regions of the Na,K-ATPase alpha-subunit and a sarcoplasmic reticulum Ca(2+)-ATPase were constructed and expressed together with the avian Na,K-ATPase beta-subunit cDNA in COS-1 cells to determine which regions of the alpha-subunit are required for assembly with the beta-subunit. Assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the avian beta-subunit. A chimera composed of the amino-terminal two-thirds of the Na,K-ATPase and carboxyl-terminal one-third of the Ca(2+)-ATPase did not assemble with the avian beta-subunit. In contrast, the reciprocal chimera, containing the carboxyl-terminal one-third of the Na,K-ATPase, assembled with the beta-subunit. A third chimera, in which 161 amino acids of the Na,K-ATPase carboxyl terminus replaced the corresponding amino acids of the Ca(2+)-ATPase carboxyl terminus, also assembled with the beta-subunit. These results suggest that the aminoacyl residues of the Na,K-ATPase alpha-subunit critical for subunit assembly lie within the carboxyl-terminal 16% of the sequence.     

Cytoplasmic and transmembrane domain deletions of Na,K-ATPase beta-subunit. Effects on subunit assembly and intracellular transport.

cDNAs encoding Na,K-ATPase beta-subunits containing deletions in the cytoplasmic domain or in the single membrane-spanning domain of the molecule were constructed and expressed in mouse L cells to determine the effect(s) of deletions in these domains on alpha/beta-subunit assembly and intracellular targeting. Avian beta-subunits lacking some or all of the cytoplasmic domain (endodomain) assemble with the endogenous mouse alpha-subunit and are correctly transported to the plasma membrane. Mutants containing deletions in the transmembrane domain were constructed by fusing portions of cDNAs encoding the amino-terminal one-third of human beta-subunit deletion mutants with avian beta-subunit cDNA encoding the carboxyl two-thirds of the molecule. A deletion of 3 amino acids in transmembrane domain resulted in correct alpha/beta-subunit assembly and localization to the plasma membrane. In contrast, deletions of 5 or more amino acids in the transmembrane domain prevented expression of the beta-subunit at the cell surface and resulted in the accumulation of these molecules in the ER. In spite of these targeting differences, all beta-subunit mutants capable of membrane insertion were also able to assemble with the alpha-subunit. These results suggest that the specificity for alpha/beta assembly resides in the ectodomains of the subunits.