F-box proteins that contain sugar-binding domains

F-box proteins are the substrate-recognition subunits of the SCF (Skp1-Cul1-F-box protein) complex, which is the largest known class of E3 ubiquitin ligases. They play important roles in ubiquitin-dependent proteolysis in eukaryotes. The human genome contains about 70 genes for F-box proteins, and at least five homologous F-box proteins containing a conserved motif in their C-termini are thought to recognize sugar chain of N-linked glycoproteins. Among theses, Fbs1 and Fbs2 are perhaps involved in the endoplasmic reticulum-associated degradation pathway. In this review, I focus on the in vivo function of Fbs1 and homologous proteins, novel intracellular oligosaccharide recognition molecules involved in the quality control system.     

Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes.

Structure-function relationships in the vertebrate U4-U6 snRNP have been analysed by assaying the ability of mutant RNAs to form U4-U6 snRNPs and to function in splicing complementation in Xenopus oocytes. The mutants define three categories of domain within the RNAs. First, domains which are not essential for splicing. These include regions of U6 which have previously been implicated in the capping and transport to the nucleus of U6 RNA as well as, less surprisingly, regions of U4 and U6 which have been poorly conserved in evolution. Second, domains whose mutation reduces U4-U6 snRNP assembly or stability. This group includes mutations in both the proposed U4-U6 interaction domain, and also, in the case of U6, in a highly conserve sequence flanking stem I of the interaction domain. These mutants are all defective in splicing. Third, regions not required for U4-U6 assembly, but required for splicing complementation. This category defines domains which are likely to be required for specific contacts with other components of the splicing machinery. Combinations of mutants in the U4 and U6 interaction domain are used to show that there are not only requirements for base complementarity but also for specific sequences in these regions.     

Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly.

U4 snRNA is phylogenetically highly conserved and organized in several domains. To determine the function of each of the domains of human U4 snRNA in the multi-step process of snRNP and spliceosome assembly, we used reconstitution procedures in combination with snRNA mutagenesis. The highly conserved 5' terminal domain of U4 snRNA consists of the stem I and stem II regions that have been proposed to base pair with U6 snRNA, and the 5' stem-loop structure. We found that each of these structural elements is essential for spliceosome assembly. However, only the stem II region is required for U4-U6 interaction, and none of these elements for Sm protein binding. In contrast, the 3' terminal domain of U4 snRNA containing the Sm binding site is dispensable for both U4-U6 interaction and spliceosome assembly. Our results support an organization of the U4 snRNP into multiple functional domains, each of which acts at distinct stages of snRNP and spliceosome assembly.     

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.     

Two different late embryogenesis abundant proteins from Arabidopsis thaliana contain specific domains that inhibit Escherichia coli growth

Late embryogenesis abundant (LEA) proteins constitute a set of proteins widespread in the plant kingdom that show common physicochemical properties such as high hydrophilicity and high content of small amino acid residues such as glycine, alanine, and serine. Typically, these proteins accumulate in response to water deficit conditions imposed by the environment or during plant normal development. In this work, we show that the over-expression in Escherichia coli of proteins of the LEA 2 and the LEA 4 families from Arabidopsis thaliana leads to inhibition of bacterial growth and that this effect is dependent on discrete regions of the proteins. Our data indicate that their antimicrobial effect is achieved through their interaction with intracellular targets. The relevance of the cationic nature and the predicted structural organization of particular protein domains in this detrimental effect on the bacteria growth process is discussed.     

Genetic analysis of human Orc2 reveals specific domains that are required in vivo for assembly and nuclear localization of the origin recognition complex

Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope-tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC-(2-5) or ORC-(1-5) complex; no other combinations of ORC subunits were detected. Moreover, only ORC-(1-5) was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC-(2-5) onto chromatin. Surprisingly, recombinant Orc2 suppressed expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC-(2-5) in the nucleus. Because this suppression required only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2.     

Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing.

Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5' trimethyl guanosine (TMG) cap, ten 2'-O-methylated residues and 13 pseudouridines. At short times after injection, cellularly derived (modified) U2 but not synthetic (unmodified) U2 rescues splicing in Xenopus oocytes depleted of endogenous U2 by RNase H targeting. After prolonged reconstitution, synthetic U2 regenerates splicing activity; a correlation between the extent of U2 modification and U2 function in splicing is observed. Moreover, 5-fluorouridine-containing U2 RNA, a potent inhibitor of U2 pseudouridylation, specifically abolishes rescue by synthetic U2, while rescue by cellularly derived U2 is not affected. By creating chimeric U2 molecules in which some sequences are from cellularly derived U2 and others are from in vitro transcribed U2, we demonstrate that the functionally important modifications reside within the 27 nucleotides at the 5' end of U2. We further show that 2'-O-methylation and pseudouridylation activities reside in the nucleus and that the 5' TMG cap is not necessary for internal modification but is crucial for splicing activity. Native gel analysis reveals that unmodified U2 is not incorporated into the spliceosome. Examination of the U2 protein profile and glycerol-gradient analysis argue that U2 modifications directly contribute to conversion of the 12S to the 17S U2 snRNP particle, which is essential for spliceosome assembly.     

Claims that intermediate filaments contain F-actin are unwarranted

Examination of sectioned, embedded material showed that almost all intermediate filaments normally present in cultured chick embryo fibroblasts, IMR-33 cells, and BHK-21 cells were eliminated by trypsinization. At the same time atypical filaments of intermediate size were formed by close apposition of thin filaments. Previous observations (Buckley et al., 1978, J. Cell Biol. 78:644-652) on negatively stained and on critical-point-dried whole-cell mounts confused these different filament types and, on the basis of heavy meromyosin binding to the atypical intermediate-sized filaments, we suggested that many normal intermediate filaments contain f-actin. The present results show that this suggestion was unwarranted and we therefore retract it.     

Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly

HeLa cell nuclear splicing extracts have been prepared that are specifically and efficiently depleted of U1, U2, or U4/U6 snRNPs by antisense affinity chromatography using biotinylated 2'-OMe RNA oligonucleotides. Removal of each snRNP particle prevents pre-mRNA splicing but arrests spliceosome formation at different stages of assembly. Mixing extracts depleted for different snRNP particles restores formation of functional splicing complexes. Specific binding of factors to the 3' splice site region is still detected in snRNP-depleted extracts. Depletion of U1 snRNP impairs stable binding of U2 snRNP to the pre-mRNA branch site. This role of U1 snRNP in promoting stable preslicing complex formation is independent of the U1 snRNA-5' splice site interaction.     

Amphipathic motifs in BAR domains are essential for membrane curvature sensing

BAR (Bin/Amphiphysin/Rvs) domains and amphipathic α‐helices (AHs) are believed to be sensors of membrane curvature thus facilitating the assembly of protein complexes on curved membranes. Here, we used quantitative fluorescence microscopy to compare the binding of both motifs on single nanosized liposomes of different diameters and therefore membrane curvature. Characterization of members of the three BAR domain families showed surprisingly that the crescent‐shaped BAR dimer with its positively charged concave face is not able to sense membrane curvature. Mutagenesis on BAR domains showed that membrane curvature sensing critically depends on the N‐terminal AH and furthermore that BAR domains sense membrane curvature through hydrophobic insertion in lipid packing defects and not through electrostatics. Consequently, amphipathic motifs, such as AHs, that are often associated with BAR domains emerge as an important means for a protein to sense membrane curvature. Measurements on single liposomes allowed us to document heterogeneous binding behaviour within the ensemble and quantify the influence of liposome polydispersity on bulk membrane curvature sensing experiments. The latter results suggest that bulk liposome‐binding experiments should be interpreted with great caution.     

Ring domains are essential for GATOR2-dependent mTORC1 activation

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Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts

The composition and function of histone storage complexes of Xenopus eggs have been investigated using monoclonal antibodies. We show that core histones are contained in two distinct complexes: H2A and H2B are associated with nucleoplasmin, and H3 and H4 are associated with nuclear protein N1. Immunodepletion analyses demonstrate that both complexes are required for nucleosome core assembly by extracts in vitro, the product being a simple sum of the histones from each complex. In addition, the majority of the stored H2A is shown to be an unusual form that migrates close to the position of H3 by SDS-polyacrylamide gel electrophoresis and resembles a variant synthesized in a cell-cycle-independent manner in mammalian cells.     

The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly.

Ligand-gated ion channels are oligomeric membrane proteins in which homologous subunits specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we used a dominant-negative assay in which truncated subunits of the mouse muscle acetylcholine receptor (AChR) were coexpressed with the four wild-type subunits in transfected COS cells. Fragments of the alpha, delta, and gamma subunits consisting solely of the extracellular N-terminal domain blocked surface expression of the AChR and the formation of alpha delta heterodimers, an early step in the assembly pathway of the AChR. Immunoprecipitation and sucrose gradient sedimentation experiments showed that an N-terminal fragment of the alpha subunit forms a specific complex with the intact delta subunit. Thus the extracellular N-terminal domain of the alpha, delta, and gamma subunits contains the information necessary for specific subunit association.     

Capnocytophaga spp. contain sulfonolipids that are novel in procaryotes.

A group of unusual sulfonolipids was found in bacteria of the genus Capnocytophaga. One of these lipids, to which we have assigned the trivial name capnine, was isolated in 98% pure form and was identified, by infrared absorption spectrometry, high-resolution mass spectrometry, and other methods, as 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid. Another lipid appears to be an N-acylated version of capnine; after acid hydrolysis, its sulfur was recovered in a form chromatographically indistinguishable from that of capnine. The new lipids are related structurally to sphingosine and the ceramides, respectively, but differ markedly from those compounds in important respects, notably the presence of the sulfonate group. Some Capnocytophaga strains accumulated mostly capnine, whereas others accumulated mostly N-acylcapnine. All seven strains examined were found to contain the new lipids, in amounts ranging from 7 to 16 mumol/g of cells (wet weight). The lipids were found in isolated cell envelopes, where they were present in amounts ranging up to 400 mg/g of envelope protein; they are, accordingly, major cell components.     

Golgi-derived membranes that contain an acylated viral polypeptide are used for vaccinia virus envelopment

A 37,000-dalton polypeptide (p37K) present on purified extracellular vaccinia virus but absent from intracellular virus particles of classical morphology (G. Hiller et al., J. Virol. 39:903-913, 1981; L. G. Payne, J. Virol. 27:28-37, 1978) was further characterized. The polypeptide was only expressed in infected cells after onset of viral DNA replication. Phase partition experiments showed that it is relatively hydrophobic. Although p37K apparently is not a glycoprotein, in vivo radioisotope labeling detected tightly associated palmitic acid. Antibodies to p37K were used to monitor its distribution within infected cells at the light and electron microscopic levels. After synthesis p37K first accumulated in the Golgi region due to a tight membrane association. During progressing infection p37K-carrying membranes were used to form double-walled envelopes around brick-shaped vaccinia particles. Within these specialized vesicles vaccinia particles were moved through the cytoplasm toward the cell's surface, presumably along cellular routes for certain secretory products. Finally, single enveloped viruses were released into the extracellular space by an exocytotic process.     

The 5'-untranslated regions of picornavirus RNAs contain independent functional domains essential for RNA replication and translation.

The role of the 5'-untranslated region (5'UTR) in the replication of enteroviruses has been studied by using a series of poliovirus type 3 (PV3) replicons containing the chloramphenicol acetyltransferase reporter gene in which the 5'UTR was replaced by the 5'UTR of either coxsackievirus B4 or human rhinovirus 14 or composite 5'UTRs derived from sequences of PV3, human rhinovirus 14, coxsackievirus B4, or encephalomyocarditis virus. The results indicate that efficient replication of an enterovirus genome requires a compatible interaction between the 5'-terminal cloverleaf structure and the coding and/or 3'-noncoding regions of the genome. A crucial determinant of this interaction is the stem-loop formed by nucleotides 46 to 81 (stem-loop d). The independence of the cloverleaf structure formed by the 5'-terminal 88 nucleotides and the ribosome landing pad or internal ribosome entry site (IRES) was investigated by constructing a 5'UTR composed of the PV3 cloverleaf and the IRES from encephalomyocarditis virus. Chloramphenicol acetyltransferase gene-containing replicons and viruses containing this recombinant 5'UTR showed levels of replication similar to those of the corresponding genomes containing the complete PV3 5'UTR, indicating that the cloverleaf and the IRES may be regarded as functionally independent and nonoverlapping elements.     

Identification of distinct c-terminal domains of the Bombyx adipokinetic hormone receptor that are essential for receptor export, phosphorylation and internalization

Neuropeptides of the adipokinetic hormone (AKH) family play important roles in insect hemolymph sugar homeostasis, larval lipolysis and storage-fat mobilization. Our previous studies have shown that the adipokinetic hormone receptor (AKHR), a Gs-coupled receptor, induces intracellular cAMP accumulation, calcium mobilization and ERK1/2 phosphorylation upon agonist stimulation. However, the underlying molecular mechanisms that regulate the internalization and desensitization of AKHR remain largely unknown. In the current study we made a construct to express AKHR fused with enhanced green fluorescent protein (EGFP) at its C-terminal end to further characterize AKHR internalization. In stable AKHR-EGFP-expressing HEK-293 cells, AKHR-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner via the clathrin-coated pit pathway upon agonist stimulation, and internalized receptors were slowly recovered to the cell surface after the removal of AKH peptides. The results derived from RNA interference and arrestin translocation demonstrated that G protein-coupled receptor kinase 2 and 5 (GRK2/5) and β-arrestin2 were involved in receptor phosphorylation and internalization. Furthermore, experiments using deletion and site-directed mutagenesis strategies identified the three residues (Thr356, Ser359 and Thr362) responsible for GRK-mediated phosphorylation and internalization and the C-terminal domain from residue-322 to residue-342 responsible for receptor export from ER. This is the first detailed investigation of the internalization and trafficking of insect G protein-coupled receptors.