Glucose and type 2A protein phosphatase regulate the interaction between catalytic and regulatory subunits of AMP-activated protein kinase

We have expressed in yeast the different subunits of AMP-activated protein kinase (AMPK) and, by using the two-hybrid system, we have found a glucose-regulated interaction between alpha 2 catalytic and gamma 1 regulatory subunits. This regulation was not affected by known regulators of the corresponding yeast orthologue, the SNF1 complex, such as Reg1 or Hxk2, but it was affected by deletion of regulatory subunits of yeast type 2A protein phosphatase (PP2A) complex. We have also found that Tpd3 and PR65 alpha, the corresponding yeast and mammalian A subunits of PP2A, interacted with AMPK alpha 2 both in yeast and mammals, respectively. This interaction occurred only through the regulatory domain of this subunit. These results suggested a direct involvement of PP2A complex in regulating the interaction between AMPK alpha 2 and gamma 1 in a glucose-dependent manner.     

A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels

Proper ion channel function often requires specific combinations of pore-forming alpha and regulatory beta subunits, but little is known about the mechanisms that regulate the surface expression of different channel combinations. Our studies of ATP-sensitive K+ channel (K(ATP)) trafficking reveal an essential quality control function for a trafficking motif present in each of the alpha (Kir6.1/2) and beta (SUR1) subunits of the K(ATP) complex. We show that this novel motif for endoplasmic reticulum (ER) retention/retrieval is required at multiple stages of K(ATP) assembly to restrict surface expression to fully assembled and correctly regulated octameric channels. We conclude that exposure of a three amino acid motif (RKR) can explain how assembly of an ion channel complex is coupled to intracellular trafficking.     

Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster.

A very large cis-regulatory region of approximately 300 kb is responsible for the complex patterns of expression of the three homeotic genes of the bithorax complex Ubx, abd-A and Abd-B. This region can be subdivided in nine parasegment-specific regulatory subunits. Recent genetic and molecular analysis has revealed the existence of two novel cis-regulatory elements Mcp and Fab-7. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B in parasegments 9 and 10. Similarly, Fab-7 is located between iab-6 and iab-7, the parasegment 11 and 12-specific regulatory units. Mcp and Fab-7 appear to function as domain boundaries that separate adjacent cis-regulatory units. We report the analysis of two new Mcp mutant deletions (McpH27 and McpB116) that allow us to localize sequences essential for boundary function to a approximately 0.4 kb DNA segment. These essential sequences closely coincide to a approximately 0.3 kb nuclease hypersensitive region in chromatin. We also show that sequences contributing to the Fab-7 boundary appear to be spread over a larger stretch of DNA, but like Mcp have an unusual chromatin structure.     

In vivo analysis of DNase I hypersensitive sites in the human CFTR gene.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex pattern of expression. The regulatory elements conferring tissue-specific and temporal regulation are thought to lie mainly outside the promoter region. Previously, we identified DNase I hypersensitive sites (DHS) that may contain regulatory elements associated with the CFTR gene at -79.5 and at -20.5 kb with respect to the ATG and at 10 kb into the first intron. MATERIALS AND METHODS: In order to evaluate these regulatory elements in vivo we examined these DHS in a human CFTR gene that was introduced on a yeast artificial chromosome (YAC) into transgenic mice. The 310 kb human CFTR YAC was shown to restore the pheno-type of CF-null mice and so is likely to contain most of the regulatory elements required for tissue-specific expression of CFTR. RESULTS: We found that the YAC does not include the -79.5 kb region. The DHS at -20.5 kb is present in the chromatin of most tissues of the transgenic mice, supporting its non-tissue-specific nature. The DHS in the first intron is present in a more restricted set of tissues in the mice, although its presence does not show complete concordance with CFTR expression. The intron I DHS may be important for the higher levels of expression found in human pancreatic ducts and in lung submucosal glands. CONCLUSION: These data support the in vivo importance of these regulatory elements.     

26S proteasome subunits are O-linked N-acetylglucosamine-modified in Drosophila melanogaster

The O-linked glycosylation of highly purified Drosophila 26S proteasome has been analyzed by immunological and lectin-binding methods. Five regulatory complex subunits and at least nine catalytic core subunits were recognized by two different monoclonal antibodies specific for O-linked N-acetylglucosamine-modified proteins, and by wheat germ agglutinin, which is specific for the N-acetylglucosamine sugar side-chain. The specificity of these reactions has been proved by competition studies with free N-acetylglucosamine. Three ATPase subunits of the regulatory complex, which are O-glycosylated, have previously been shown [FEBS Lett. 430 (1998) 269] to occur in phosphorylated form as well, indicating that several different post-translational modifications, with distinct regulatory potential, may be present on the same subunit.     

The sheep genome contributes to localization of control elements in a human gene with complex regulatory mechanisms

Genes that show complex tissue-specific and temporal control by regulatory elements located outside their promoters present a considerable challenge to identify the sequences involved. The rapid accumulation of genomic sequence information for a number of species has enabled a comparative phylogenetic approach to find important regulatory elements. For some genes, which show a similar pattern of expression in humans and rodents, genomic sequence information for these two species may be sufficient. Others, such as the cystic fibrosis transmembrane conductance regulator (CFTR) gene, show significant divergence in expression patterns between mouse and human, necessitating phylogenetic approaches involving additional species. The ovine CFTR gene has a temporal and spatial expression pattern that is very similar to that of human CFTR. Comparative genomic sequence analysis of ovine and human CFTR identified high levels of homology between the core elements in several potential regulatory elements defined as DNase I hypersensitive sites in human CFTR. These data provide a case for the power of an artiodactyl genome to contribute to the understanding of human genetic disease.     

Maize protein kinase CK2: regulation and functionality of three beta regulatory subunits

Biochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2alpha), thus lacking CK2beta regulatory subunits. To investigate the existence and functionality of CK2beta regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three full-length cDNAs encoding CK2beta regulatory subunits (CK2beta-1, CK2beta-2 and CK2beta-3) and a cDNA coding for a novel CK2alpha catalytic subunit, CK2alpha-3. The pattern of expression of all these alpha/beta subunits has been studied in different organs and developmental stages using specific probes for each isoform, and indicates that while CK2alpha subunits are constitutive, CK2beta subunits are expressed differentially during embryo development. The yeast two-hybrid system and pull-down assays have been used to study specific interactions between the different subunits. While CK2alpha subunits are unable to self-associate, preferential interactions between alpha/beta isoforms and beta/beta isoforms can be predicted. Furthermore, we show that maize CK2alpha/beta subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2beta subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2beta regulatory subunits.     

Regulatory elements controlling expression of the Drosophila homeotic gene fork head.

The region-specific homeotic gene fork head (fkh) is expressed and required in a variety of tissues of the developing Drosophila embryo. In order to identify the cis regulatory elements directing the complex spatio-temporal expression pattern of fkh, we have studied the subpatterns directed by defined fragments of fkh genomic DNA. These experiments enabled us to distinguish separate regulatory elements specific for the different expression domains of fkh. In addition, our analysis revealed several unexpected features such as the redundancy of regulatory elements and the overlap of regulatory elements with the transcribed regions of other genes. Moreover, the separation of normally contiguous elements effecting expression in the posterior terminal fkh domain appears to lead to novel expression domains which do not correspond to known developmental units in the embryo.     

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha–beta–PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA–PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha–alpha and alpha–PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha–alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)₂.     

Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly regulated cellular process

Cytochrome c-oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in the regulation of aerobic production of energy. Biogenesis of eukaryotic COX involves the coordinated action of two genomes. Three mitochondrial DNA-encoded subunits form the catalytic core of the enzyme, which contains metal prosthetic groups. Another 10 subunits encoded in the nuclear DNA act as a protective shield surrounding the core. COX biogenesis requires the assistance of >20 additional nuclear-encoded factors acting at all levels of the process. Expression of the mitochondrial-encoded subunits, expression and import of the nuclear-encoded subunits, insertion of the structural subunits into the mitochondrial inner membrane, addition of prosthetic groups, assembly of the holoenzyme, further maturation to form a dimer, and additional assembly into supercomplexes are all tightly regulated processes in a nuclear-mitochondrial-coordinated fashion. Such regulation ensures the building of a highly efficient machine able to catalyze the safe transfer of electrons from cytochrome c to molecular oxygen and ultimately facilitate the aerobic production of ATP. In this review, we will focus on describing and analyzing the present knowledge about the different regulatory checkpoints in COX assembly and the dynamic relationships between the different factors involved in the process. We have used information mostly obtained from the suitable yeast model, but also from bacterial and animal systems, by means of large-scale genetic, molecular biology, and physiological approaches and by integrating information concerning individual elements into a cellular system network.     

细菌转录起始调控机制*

原核生物同一种群的每个细胞都是和外界环境直接接触的,它们主要通过开启或关闭某些基因的表达来适应环境条件。所以,环境因子往往是调控的效应因子,必须严格调控转录来确保细胞对环境改变做出有效且充分的反应。原核生物基因的表达受多种因素的调控,而对于大多数细菌来说,调控基因表达的关键步骤是启动子识别和RNA聚合酶启动转录。在细菌的细胞中,可以通过调节RNA聚合酶的活性以及改变RNA聚合酶对启动子的结合来优化基因的转录过程以适应不同环境变化。总结了目前已发现的参与细菌细胞转录调节的各类因子,从这些因子对启动子的作用、RNA聚合酶的作用以及两者的相互作用等方面阐述它们调控基因表达的分子机制。总结多种基因调控的作用,加深对转录起始过程的认识,希望能对未来调控转录起始过程来实现目标基因的高效表达和不利基因的抑制表达提供思路,为以后的工业菌株改造提供依据。