SmaI Site Research Articles

Structures of the Carbohydrate Recognition Domain of Ca2+-independent Cargo Receptors Emp46p and Emp47p

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

Importin-β Mediates Cdc7 Nuclear Import by Binding to the Kinase Insert II Domain, Which Can Be Antagonized by Importin-α

We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-beta binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-alpha and -beta bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-beta is approximately 10 times higher than for importin-alpha at low protein concentrations of an equimolar ratio. Immunodepletion of importin-beta, but not importin-alpha, abrogates Cdc7 nuclear import, and the addition of importin-beta to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-beta, but not anti-importin-alpha antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-alpha to Cdc7. Further studies by site-directed mutagenesis suggest that Lys306 and Lys309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization.

Pir Proteins of Saccharomyces cerevisiae Are Attached to β-1,3-Glucan by a New Protein-Carbohydrate Linkage

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

PTEN Deletion Leads to Up-regulation of a Secreted Growth Factor Pleiotrophin

Tumor suppressor gene PTEN is highly mutated in a wide variety of human tumors. To identify unknown targets or signal transduction pathways that are regulated by PTEN, microarray analysis was performed to compare the gene expression profiles of Pten null mouse embryonic fibroblasts (MEFs) cell lines and their isogenic counterparts. Expression of a heparin binding growth factor, pleiotrophin (Ptn), was found to be up-regulated in Pten-/- MEFs as well as Pten null mammary tumors. Further experiments revealed that Ptn expression is regulated by the PTEN-PI3K-AKT pathway. Knocking down the expression of Ptn by small interfering RNA resulted in the reduction of Akt and GSK-3beta phosphorylation and suppression of the growth and the tumorigenicity of Pten null MEFs. Our results suggest that PTN participates in tumorigenesis caused by PTEN loss and PTN may be a potential target for anticancer therapy, especially for those tumors with PTEN deficiencies.

Nuclear Transportation of Diacylglycerol Kinase γ and Its Possible Function in the Nucleus

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGKgamma came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGKgamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKgamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKgamma acts as a nuclear transport signal. Furthermore, to address the function of DGKgamma in the nucleus, we produced stable cell lines of wild-type DGKgamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKgamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKgamma regulates cell cycle.

Growth Phase-dependent Activation of Nitrogen-related Genes by a Control Network of Group 1 and Group 2 σ Factors in a Cyanobacterium

It has been reported that an RNA polymerase sigma factor, SigC, mainly contributes to specific transcription from the promoter PglnB-54,-53 under nitrogen-deprived conditions during the stationary phase of cell growth in the cyanobacterium Synechocystis sp. strain PCC 6803 (Asayama, M., Imamura, S., Yoshihara, S., Miyazaki, A., Yoshida, N., Sazuka, T., Kaneko, T., Ohara, O., Tabata, S., Osanai, T., Tanaka, K., Takahashi, H., and Shirai, M. (2004) Biosci. Biotechnol. Biochem. 68, 477-487). In this study, we further examined the functions of group 2 sigma factors of RNA polymerase in NtcA-dependent nitrogen-related gene expression in PCC 6803. Results indicated that SigB and SigC contribute to the transcription from PglnB-54,-53 with a sigma factor replaced in a growth phase-dependent manner. We also confirmed the contribution of SigB and SigC to the transcription of other NtcA-dependent genes, glnA, sigE, and amt1, as in the case of glnB. On the other hand, the transcription of glnN was dependent on SigB and SigE. In the SigB and SigC-based regulation, the level of SigB increased, but that of SigC was constant under conditions of nitrogen deprivation. Furthermore, it was found that SigC negatively and positively regulates the level of SigB in the log and stationary phase, respectively. SigC also had a positive effect on the level of sigB transcript during the stationary phase. In contrast, SigB acts positively on SigC levels in both growth phases. These results and previous findings indicated that multiple group 2 sigma factors take part in the control of NtcA-dependent nitrogen-related gene expression in cooperation with a group 1 sigma factor, SigA.

The C-terminal Activating Region 2 of the Epstein-Barr Virus-encoded Latent Membrane Protein 1 Activates NF-κB through TRAF6 and TAK1

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for EBV-mediated B cell transformation. LMP1 is capable of activating several intracellular signaling pathways including the NF-kappaB pathway, which contributes to the EBV-mediated cell transformation. Two regions in the cytoplasmic carboxyl tail of LMP1, namely C-terminal activating regions 1 and 2 (CTAR1 and CTAR2), are responsible for NF-kappaB activation, with CTAR2 being the main NF-kappaB activator. Although the CTAR1-mediated NF-kappaB activation was previously shown to be TRAF3-dependent, we showed here that the CTAR2-mediated NF-kappaB activation is mainly TRAF6-dependent but TRAF2/5-independent. In contrast to the interleukin-1 receptor/toll-like receptor-mediated NF-kappaB pathways, the CTAR2-mediated NF-kappaB pathway does not require MyD88, IRAK1, or IRAK4 for TRAF6 engagement. Furthermore, we showed that TAK1 is required for NF-kappaB activation by LMP1. Thus, LMP1 utilizes two distinct pathways to activate NF-kappaB: a major one through CTAR2/TRAF6/TAK1/IKKbeta (canonical pathway) and a minor one through CTAR1/TRAF3/NIK/IKKalpha (noncanonical pathway).

Control of MYEOV Protein Synthesis by Upstream Open Reading Frames

The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5'-untranslated region (5'-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5'-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5'-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5'-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter.

Mediator as a General Transcription Factor

Others have shown that yeast strains bearing a ts mutation in the Srb4 subunit of Mediator cease transcription of all mRNA at the restrictive temperature, in a manner virtually indistinguishable from a strain bearing a ts mutation in the largest subunit of RNA polymerase II. We find that srb4ts Mediator is defective for the stimulation of basal RNA polymerase II transcription at the restrictive temperature in vitro. Taken together, these findings lead to the suggestion that Mediator is required for basal RNA polymerase II transcription in vivo. On this basis, Mediator is identified as a general transcription factor, comparable in importance to RNA polymerase II and other general factors for the initiation of transcription. The possibility that Mediator serves as an anti-inhibitor, opposing the effects of global negative regulators, is largely excluded.

Proliferating Cell Nuclear Antigen Recruits Cyclin-dependent Kinase Inhibitor Xic1 to DNA and Couples Its Proteolysis to DNA Polymerase Switching

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.

Dlx5 Specifically Regulates Runx2 Type II Expression by Binding to Homeodomain-response Elements in the Runx2 Distal Promoter

Two major isoforms of the Runx2 gene are expressed by alternative promoter usage: Runx2 type I (Runx2-I) is derived from the proximal promoter (P2), and Runx2 type II (Runx2-II) is produced by the distal promoter (P1). Our previous results indicate that Dlx5 mediates BMP-2-induced Runx2 expression and osteoblast differentiation (Lee, M.-H., Kim, Y-J., Kim, H-J., Park, H-D., Kang, A-R., Kyung, H.-M., Sung, J-H., Wozney, J. M., Kim, H-J., and Ryoo, H-M. (2003) J. Biol. Chem. 278, 34387-34394). However, little is known of the molecular mechanisms by which Dlx5 up-regulates Runx2 expression in BMP-2 signaling. Here, Runx2-II expression was found to be specifically stimulated by BMP-2 treatment or by Dlx5 overexpression. In addition, BMP-2, Dlx5, and Runx2-II were found to be expressed in osteogenic fronts and parietal bones of the developing cranial vault and Runx2-I and Msx2 in the sutural mesenchyme. Furthermore, Runx2 P1 promoter activity was strongly stimulated by Dlx5 overexpression, whereas Runx2 P2 promoter activity was not. Runx2 P1 promoter deletion analysis indicated that the Dlx5-specific response is due to sequences between -756 and -342 bp of the P1 promoter, where three Dlx5-response elements are located. Dlx5 responsiveness to these elements was confirmed by gel mobility shift assay and site-directed mutagenesis. Moreover, Msx2 specifically suppressed the Runx2 P1 promoter, and the responsible region overlaps with that recognized by Dlx5. In summary, Dlx5 specifically transactivates the Runx2 P1 promoter, and its action on the P1 promoter is antagonized by Msx2.

Crystal Structure of the Yersinia enterocolitica Type III Secretion Chaperone SycT

Several Gram-negative pathogens deploy type III secretion systems (TTSSs) as molecular syringes to inject effector proteins into host cells. Prior to secretion, some of these effectors are accompanied by specific type III secretion chaperones. The Yersinia enterocolitica TTSS chaperone SycT escorts the effector YopT, a cysteine protease that inactivates the small GTPase RhoA of targeted host cells. We solved the crystal structure of SycT at 2.5 angstroms resolution. Despite limited sequence similarity among TTSS chaperones, the SycT structure revealed a global fold similar to that exhibited by other structurally solved TTSS chaperones. The dimerization domain of SycT, however, differed from that of all other known TTSS chaperone structures. Thus, the dimerization domain of TTSS chaperones does not likely serve as a general recognition pattern for downstream processing of effector/chaperone complexes. Yersinia Yop effectors are bound to their specific Syc chaperones close to the Yop N termini, distinct from their catalytic domains. Here, we showed that the catalytically inactive YopT(C139S) is reduced in its ability to bind SycT, suggesting an ancillary interaction between YopT and SycT. This interaction could maintain the protease inactive prior to secretion or could influence the secretion competence and folding of YopT.

Phenotypic and Biochemical Analyses of BACE1- and BACE2-deficient Mice

Beta-secretase (BACE1) is the rate-limiting protease for the generation of the amyloid beta-peptide (Abeta) in Alzheimer disease. Mice in which the bace1 gene is inactivated are reported to be healthy. However, the presence of a homologous gene encoding BACE2 raises the possibility of compensatory mechanisms. Therefore, we have generated bace1, bace2, and double knockout mice. We report here that BACE1 mice display a complex phenotype. A variable but significant number of BACE1 offspring died in the first weeks after birth. The surviving mice remained smaller than their littermate controls and presented a hyperactive behavior. Electrophysiologically, subtle alterations in the steady-state inactivation of voltage-gated sodium channels in BACE1-deficient neurons were observed. In contrast, bace2 knockout mice displayed an overall healthy phenotype. However, a combined deficiency of BACE2 and BACE1 enhanced the bace1-/- lethality phenotype. At the biochemical level, we have confirmed that BACE1 deficiency results in an almost complete block of Abeta generation in neurons, but not in glia. As glia are 10 times more abundant in brain compared with neurons, our data indicate that BACE2 could indeed contribute to Abeta generation in the brains of Alzheimer disease and, in particular, Down syndrome patients. In conclusion, our data challenge the general idea of BACE1 as a safe drug target and call for some caution when claiming that no major side effects should be expected from blocking BACE1 activity.

Deletion of Cg-emb in Corynebacterianeae Leads to a Novel Truncated Cell Wall Arabinogalactan, whereas Inactivation of Cg-ubiA Results in an Arabinan-deficient Mutant with a Cell Wall Galactan Core

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide.

Functional Characterization of the Iron-regulatory Transcription Factor Fep1 from Schizosaccharomyces pombe

In response to excess iron, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in iron uptake through the Fep1 transcription factor. Fep1 mediates this control by interacting with the consensus sequence 5'-(A/T)GATAA-3', found in iron-dependent promoters. In this report, we show that Fep1 localizes to the nucleus under both iron-replete and iron-starved conditions. The Fep1 DNA binding domain (amino acids 1-241) contains two GATA-type zinc finger motifs. Although we determine that the Fep1 C-terminal zinc finger (ZF2) is essential for DNA binding, we show that the N-terminal zinc finger (ZF1) enhances DNA binding affinity approximately 5-fold. Between the two zinc finger motifs of Fep1 resides an invariant amino acid sequence, denoted the Cys-rich region (amino acids 68-94), in which four highly conserved Cys residues are found. Cells harboring mutant alleles in which two or more of the conserved Cys residues were substituted by alanine exhibited elevated fio1(+) mRNA levels. We determine that the dissociation constant for the resulting complex between each of the Cys mutants and the sequence 5'-(A/T)GATAA-3' reflects a much lower affinity that correlates with failure to repress fio1(+) gene expression. Deletion analysis identified two heptad repeats (amino acids 522-536) within the C-terminal region of Fep1 that are necessary and sufficient to mediate Fep1 dimerization. Moreover, mutations that impair dimerization also negatively affect transcriptional repression. Together these findings reveal several novel features of Fep1, a non-canonical GATA factor required for iron homeostasis.

The Crucial Role of Trehalose and Structurally Related Oligosaccharides in the Biosynthesis and Transfer of Mycolic Acids in Corynebacterineae

Trehalose (alpha-D-glucopyranosyl-alpha'-D-glucopyranoside) is essential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the so-called Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on alpha-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.

High Mobility Group Protein-B1 Interacts with Sterol Regulatory Element-binding Proteins to Enhance Their DNA Binding

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that are predominately involved in the regulation of lipogenic and cholesterogenic enzyme gene expression. To identify unknown proteins that interact with SREBP, we screened nuclear extract proteins with 35S-labeled SREBP-1 bait in Far Western blotting analysis. Using this approach, high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel SREBP interacting protein. In vitro glutathione S-transferase pull-down and in vivo coimmunoprecipitation studies confirmed an interaction between HMGB1 and both SREBP-1 and -2. The protein-protein interaction was mediated through the helix-loop-helix domain of SREBP-1, residues 309-344, and the A box of HMGB1. Furthermore, an electrophoretic mobility shift assay demonstrated that HMGB1 enhances SREBPs binding to their cognate DNA sequences. Moreover, luciferase reporter analyses, including RNA interference technique showed that HMGB1 potentiates the transcriptional activities of SREBP in cultured cells. These findings raise the intriguing possibility that HMGB1 is potentially involved in the regulation of lipogenic and cholesterogenic gene transcription.

Epigenetic silencing of TCEAL7 (Bex4) in ovarian cancer

Epigenetic silencing by hypermethylation of CpGs represents a mechanism of inactivation of tumor suppressors. Here we report on the cloning of a novel candidate tumor suppressor gene TCEAL7 inactivated by methylation in ovarian cancer. TCEAL codes for a 1.35 kb transcript that was previously reported to be downregulated in ovarian cancer by cDNA microarray and suppression subtraction cDNA (SSH) analyses. This report focuses on the elucidation of mechanisms associated with TCEAL7 downregulation. Expression of TCEAL7 is downregulated in a majority of ovarian tumors and cancer cell lines but induced by 5-aza-2'-deoxycytidine treatment in a dose-dependant manner, implicating methylation as a mechanism of TCEAL7 inactivation. Sequence analyses of bisufite-modified genomic DNA from somatic cell hybrids with either the active or the inactive human X chromosome reveal that TCEAL7 is subjected to X chromosome inactivation. Loss of TCEAL7 expression in primary tumors and cell lines correlates with methylation of a CpG site within the promoter. In vitro methylation of the CpG site suppresses promoter activity whereas selective demethylation of the SmaI site attenuates the suppression. Finally, re-expression of TCEAL7 in cancer cell lines induces cell death and reduces colony formation efficiency. These data implicate TCEAL7 as a cell death regulatory protein that is frequently inactivated in ovarian cancers, and suggest that it may function as a tumor suppressor.

Membrane Topology of the STT3 Subunit of the Oligosaccharyl Transferase Complex

The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N(cyt)-C(lum) orientation.

Importin α/β Mediates Nuclear Transport of a Mammalian Circadian Clock Component, mCRY2, Together with mPER2, through a Bipartite Nuclear Localization Signal

Circadian rhythms, which period is approximately one day, are generated by endogenous biological clocks. These clocks are found throughout the animal kingdom, as well as in plants and even in prokaryotes. Molecular mechanisms for circadian rhythms are based on transcriptional oscillation of clock component genes, consisting of interwoven autoregulatory feedback loops. Among the loops, the nuclear transport of clock proteins is a crucial step for transcriptional regulation. In the present study, we showed that the nuclear entry of mCRY2, a mammalian clock component, is mediated by the importin alpha/beta system through a bipartite nuclear localization signal in its carboxyl end. In vitro transport assay using digitonin-permeabilized cells demonstrated that all three importin alphas, alpha1 (Rch1), alpha3 (Qip-1), and alpha7 (NPI-2), can mediate mCRY2 import. mCRY2 with the mutant nuclear localization signal failed to transport mPER2 into the nucleus of mammalian cultured cells, indicating that the nuclear localization signal identified in mCRY2 is physiologically significant. These results suggest that the importin alpha/beta system is involved in nuclear entry of mammalian clock components, which is indispensable to transcriptional oscillation of clock genes.