Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter: ROLE OF LYSINE 63-LINKED UBIQUITIN CHAINS*

Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys⁶³ linked chain(s), and Jen1-Lys³³⁸ is one of the target residues. Ubiquitin-Lys⁶³-linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys⁶³-linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.Ubiquitylation is one of the most prevalent protein post-translational modifications in eukaryotes. In addition to its role in promoting proteasomal degradation of target proteins, ubiquitylation has been shown to regulate multiple processes, including DNA repair, signaling, and intracellular trafficking. Ubiquitylation serves as a key signal mediating the internalization of plasma membrane receptors and transporters, followed by their intracellular transport and subsequent recycling or lysosomal/vacuolar degradation (1, 2). In Saccharomyces cerevisiae, transporters usually display both constitutive and accelerated endocytosis regulated by factors such as excess substrate, changes in nutrient availability, and stress conditions. Ubiquitylation of these cell surface proteins acts as a signal triggering their internalization (1). A single essential E3⁴ ubiquitin ligase, Rsp5, has been implicated in the internalization of most, if not all, endocytosed proteins (3). Rsp5 is the unique member in S. cerevisiae of the HECT (homologous to E6AP COOH terminus)-ubiquitin ligases of the Nedd4/Rsp5 family (4). In a few cases, Rsp5-dependent cell surface ubiquitylation was shown to involve PY-containing adapters that bind to Rsp5 (5–7). Rsp5-mediated ubiquitylation is also required for sorting into multivesicular bodies (MVBs) of endosomal membrane proteins that come from either the plasma membrane (through endocytosis) or the Golgi (through vacuolar protein sorting (VPS) pathway) (8). Although much progress has been made in elucidating the mechanistic basis of various steps in protein trafficking, the precise requirement for a specific type and length of Ub chains at various stages of the endocytic pathway remains to be addressed.The ubiquitin profile needed for proper internalization has been established for some yeast membrane proteins (1). The α-factor receptor Ste2 was described as undergoing monoubiquitylation on several lysines (multimonoubiquitylation). The a-factor receptor, Ste3p; the general transporter of amino acids, Gap1; the zinc transporter, Ztr1; and the uracil transporter, Fur4, have been shown to be modified by short chains of two to three ubiquitins, each attached to one, two, or more target lysine residues (oligo-ubiquitylation). Among them, Fur4 and Gap1 were the only transporters demonstrated to undergo plasma membrane oligo-ubiquitylation with ubiquitin residues linked via ubiquitin-Lys⁶³ (9, 10). In addition, the two siderophore transporters Arn1 and Sit1 were also shown to undergo Lys⁶³-linked cell surface ubiquitylation (11, 12). Whether these four transporters are representative of a larger class of plasma membrane substrates remains to be determined. Little is known about the type of ubiquitylation involved and/or required for sorting to MVBs. Some MVB cargoes appear to undergo monoubiquitylation (8), whereas Sna3, an MVB cargo of unknown function, undergoes Lys⁶³-linked ubiquitylation (13). Lys⁶³-linked ubiquitin chains were also recently reported to be required, directly or indirectly, for MVB sorting of the siderophore transporter, Sit1, when trafficking through the VPS pathway in the absence of its external substrate (11). In agreement with the possibility that additional membrane-bound proteins might undergo Lys⁶³-linked ubiquitylation, a proteomic study aiming to uncover ubiquitylated yeast proteins showed that Lys⁶³-ubiquitin chains are far more abundant than previously thought (14).The transport of monocarboxylates, such as lactate and pyruvate, as well as ketone bodies across the plasma membrane is essential for the metabolism of cells of various organisms. A family of monocarboxylate transporters has been reported that includes mainly mammalian members (15). In S. cerevisiae, two monocarboxylate-proton symporters have been described, Jen1 and Ady2 (16, 17). These transporters exhibit differences in their mechanisms of regulation and specificity. Jen1 is a lactate-pyruvate-acetate-propionate transporter induced in lactic or pyruvic acid-grown cells (18). Ady2, which accepts acetate, propionate, or formate, is present in cells grown in non-fermentable carbon sources (19). Jen1 has unique regulatory characteristics and has been extensively studied. It was the first secondary porter of S. cerevisiae characterized by heterologous expression in Pichia pastoris at both the cell and the membrane vesicle levels (20). The addition of glucose to lactic acid-grown cells very rapidly triggers loss of Jen1 activity and repression of JEN1 gene expression (21, 22). Newly synthesized Jen1-GFP fusion protein is sorted to the plasma membrane in an active and stable form, and loss of Jen1-GFP activity upon glucose addition is the result of its endocytosis followed by vacuolar degradation (23). Data from large scale analyses based on mass spectrometry approaches led to the detection of two sites of ubiquitylation for Jen1, one located in the N terminus of the protein and the second in the central loop (14), and several sites of phosphorylation in the N terminus, central loop, and C terminus of the protein (14, 24). In the present study, we aimed at further characterizing the internalization step of endocytosis of the transporter Jen1 and the potential role of the phosphorylation and ubiquitylation events required for its correct endocytic trafficking.     

Endocytic Machinery Protein SlaB Is Dispensable for Polarity Establishment but Necessary for Polarity Maintenance in Hyphal Tip Cells of Aspergillus nidulans

The Aspergillus nidulans endocytic internalization protein SlaB is essential, in agreement with the key role in apical extension attributed to endocytosis. We constructed, by gene replacement, a nitrate-inducible, ammonium-repressible slaB1 allele for conditional SlaB expression. Video microscopy showed that repressed slaB1 cells are able to establish but unable to maintain a stable polarity axis, arresting growth with budding-yeast-like morphology shortly after initially normal germ tube emergence. Using green fluorescent protein (GFP)-tagged secretory v-SNARE SynA, which continuously recycles to the plasma membrane after being efficiently endocytosed, we establish that SlaB is crucial for endocytosis, although it is dispensable for the anterograde traffic of SynA and of the t-SNARE Pep12 to the plasma and vacuolar membrane, respectively. By confocal microscopy, repressed slaB1 germlings show deep plasma membrane invaginations. Ammonium-to-nitrate medium shift experiments demonstrated reversibility of the null polarity maintenance phenotype and correlation of normal apical extension with resumption of SynA endocytosis. In contrast, SlaB downregulation in hyphae that had progressed far beyond germ tube emergence led to marked polarity maintenance defects correlating with deficient SynA endocytosis. Thus, the strict correlation between abolishment of endocytosis and disability of polarity maintenance that we report here supports the view that hyphal growth requires coupling of secretion and endocytosis. However, downregulated slaB1 cells form F-actin clumps containing the actin-binding protein AbpA, and thus F-actin misregulation cannot be completely disregarded as a possible contributor to defective apical extension. Latrunculin B treatment of SlaB-downregulated tips reduced the formation of AbpA clumps without promoting growth and revealed the formation of cortical “comets” of AbpA.Germinating asexual spores (conidiospores) of Aspergillus nidulans transiently undergo isotropic growth (“swelling”) before establishing a polarity axis that grows by apical extension, leading to the characteristic tubular morphology of the fungal cell (15, 16, 33). Stable maintenance of a polarity axis at the high apical extension rates of A. nidulans (∼0.5 μm/min at 25°C) (23) can be attributable, at least in part, to the polarization of the secretory apparatus and the predominant and highly efficient delivery of secretory vesicles to the apex (8, 18, 40, 49). In addition, work from several laboratories strongly indicated that hyphal tip growth also involves endocytosis. A key observation supporting this involvement was that despite the fact that endocytosis can occur elsewhere, the endocytic internalization machinery predominates in the hyphal tip, forming a subapical collar. The spatial association of this collar with the apical region where secretory materials are delivered would allow removal of excess lipids/proteins reaching the plasma membrane with secretory vesicles (1, 2, 30, 49, 51, 57), but, most importantly, rapid endocytic recycling (i.e., efficient endocytosis of membrane proteins followed by their redelivery to the plasma membrane) can generate and maintain polarity, as shown with the v-SNARE and secretory-vesicle-resident SynA, which is a substrate of the subapical endocytic ring (1, 49, 52). It is plausible that such a mechanism could drive the polarization of one or more proteins acting as positional cues to mediate polarity maintenance.Genetic evidence strongly supported the conclusion that endocytosis is required for apical extension. Mutational inactivation of the A. nidulans fimbrin FimA or of the small GTPase ArfB^Arf6 (a regulator of endocytosis from fungi to mammals), led to delayed polarity establishment and morphologically aberrant tips indicative of polarity maintenance defects (30, 51). These mutations, although very severely debilitating, are not lethal. In contrast, heterokaryon rescue showed that SlaB, a key F-actin regulator of the endocytic internalization machinery (26), is essential in A. nidulans (2). slaBΔ cells are able to establish polarity, but they arrest in apical extension during the very early steps of polarity maintenance with a bud-like germ tube (2). However, work with Aspergillus oryzae using a thiamine-repressible promoter to drive A. oryzae End4 (AoEnd4) (SlaB) expression showed that although endocytosis was prevented and hyphal morphology altered under repressing conditions, hyphal tip extension and polarity maintenance were not completely abolished (20), perhaps suggesting that the phenotype of the thiamine-repressed conditional allele might not fully resemble the null phenotype.F-actin strongly predominates in the hyphal tips (2, 14, 17, 49, 51). Because endocytic internalization is powered by F-actin (27), predominance of endocytic “patches” (i.e., sites of endocytic internalization) in the tip accounts, at least in part, for F-actin polarization. However, F-actin plays fundamental nonendocytic roles in the tip; for example, actin cables nucleated by the SepA formin localizing to the apical crescent are thought to play a major role in secretion, whereas a network of F-actin microfilaments appears to be a major component of the Spitzenkörper (4, 21, 43, 49). Notably, all genes used to address the role of endocytosis in apical extension share in common their involvement in regulating F-actin. Thus, the Saccharomyces cerevisiae ArfB orthologue Arf3p regulates endocytosis but also appears to regulate F-actin at multiple levels (12, 28, 44). Like their respective S. cerevisiae orthologues Sla2p and Sac6p, SlaB and FimA are key components of endocytic patches, but in budding yeast their orthologues appear to regulate F-actin dynamics beyond endocytosis (27, 35, 56).To gain insight into the essential role of SlaB in A. nidulans, we designed a conditional expression allele. Time-lapse microscopy under restrictive conditions demonstrated that polarity establishment is essentially normal but that these mutant germ tubes arrested in apical extension subsequently undergo swelling, acquiring the characteristic bud-like shape of abortive slaBΔ germlings. Medium shift experiments allowed us to address the role of SlaB in apical extension beyond these early stages of polarity maintenance. We demonstrate the key role that SlaB plays in endocytosis and polarity maintenance, but we also show that deficiency of SlaB affects the actin cytoskeleton.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.The endocytosis, endocytic recycling, and endosomal sorting of numerous transport proteins and receptors are regulated by ubiquitination (1–6). Ubiquitin, an 8-kDa protein, is conjugated to target proteins via a series of steps that includes ubiquitin-activating enzymes (E1),² ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (1). Proteins that are ubiquitinated in the plasma membrane are internalized and are either deubiquitinated and recycle back to the plasma membrane or, via interactions with the endosomal sorting complexes required for transport machinery, are delivered to the lysosome for degradation (1–7). Sorting of ubiquitinated plasma membrane proteins for either the lysosomal pathway or for the recycling pathway is regulated, in part, by the removal of ubiquitin by deubiquitinating enzymes (DUBs) (1–6). Thus, the balance between ubiquitination and deubiquitination regulates the plasma membrane abundance of several membrane proteins, including the epithelial sodium channel (ENaC), the epidermal growth factor receptor, the transforming growth factor-β receptor, and the cytokine receptor γ-c (8–14).CFTR is rapidly endocytosed from the plasma membrane and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with >75% of endocytosed wild-type CFTR recycling back to the plasma membrane (15–18). A study published several years ago demonstrated that, although ubiquitination did not regulate CFTR endocytosis, ubiquitination reduced the plasma membrane abundance of CFTR in BHK cells by redirecting CFTR from recycling endosomes to lysosomes for degradation (19). However, neither the E3 ubiquitin ligase(s) responsible for the ubiquitination of CFTR nor the DUB(s) responsible for the deubiquitination of CFTR in the endocytic pathway have been identified in any cell type. Moreover, the effect of the ubiquitin status of CFTR on its endocytic sorting in human airway epithelial cells has not been reported. Thus, the goals of this study were to determine if the ubiquitin status regulates the post-endocytic sorting of CFTR in polarized airway epithelial cells, and to identify the DUBs that deubiquitinate CFTR.Approximately 100 DUBs have been identified in the human genome and are classified into five families based on sequence similarity and mechanism of action (1–6, 20, 21). To identify DUBs that regulate the deubiquitination of CFTR from this large class of enzymes, we chose an activity-based, chemical probe screening approach developed by Dr. Hidde Ploegh (4, 21, 22). This approach utilizes a hemagglutinin (HA)-tagged ubiquitin probe engineered with a C-terminal modification incorporating a thiol-reactive group that forms an irreversible, covalent bond with active DUBs. Using this approach we demonstrated in polarized human airway epithelial cells that ubiquitin-specific protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and thus its trafficking in the post-endocytic compartment. These studies demonstrate a novel function for USP10 in promoting the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Ubiquitination Regulates Proteolytic Processing of G Protein-coupled Receptors after Their Sorting to Lysosomes

Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine δ-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized δ-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor''s hydrophobic core.A fundamental cellular mechanism contributing to homeostatic regulation of receptor-mediated signal transduction involves ligand-induced endocytosis of receptors followed by proteolysis in lysosomes. The importance of such proteolytic down-regulation has been documented extensively for a number of seven-transmembrane or G protein-coupled receptors (GPCRs),³ which comprise the largest known family of signaling receptors expressed in animals, as well as for other important signaling receptors, such as the epidermal growth factor receptor tyrosine kinase (1–5).One GPCR that is well known to undergo endocytic trafficking to lysosomes is the δ-opioid peptide receptor (DOR or DOP-R) (6). Following endocytosis, DOR traffics efficiently to lysosomes in both neural and heterologous cell models (6–8), whereas many membrane proteins, including various GPCRs, recycle rapidly to the plasma membrane (9–12). Such molecular sorting of internalized receptors between divergent recycling and degradative pathways is thought to play a fundamental role in determining the functional consequences of regulated endocytosis (2, 3, 13, 14). The sorting process that directs internalized DOR to lysosomes is remarkably efficient and appears to occur rapidly (within several min) after receptor endocytosis (11). Nevertheless, biochemical mechanisms that control lysosomal trafficking and proteolysis of DOR remain poorly understood.A conserved mechanism that promotes lysosomal trafficking of a number of membrane proteins, including various signaling receptors, is mediated by covalent modification of cytoplasmic lysine residues with ubiquitin (4, 15–17). Ubiquitination was first identified as an endocytic sorting determinant in studies of vacuolar trafficking of the yeast GPCR Ste2p (18). Subsequent studies have established numerous examples of lysyl-ubiquitination being required for sorting endocytic cargo to lysosomes and have identified conserved machinery responsible for the targeting of ubiquitinated cargo to lysosomes (3, 17, 19–22).The CXCR4 chemokine receptor provides a clear example of ubiquitin-dependent lysosomal sorting of a mammalian GPCR. Ubiquitination of the carboxyl-terminal cytoplasmic domain of the CXCR4 receptor, mediated by the E3 ubiquitin ligase AIP4, is specifically required for the HRS- and VPS4-dependent trafficking of internalized receptors to lysosomes. Blocking this ubiquitination event by Lys → Arg mutation of the receptor specifically inhibits trafficking of internalized receptors to lysosomes, resulting in recycling rather than lysosomal proteolysis of receptors after ligand-induced endocytosis (23–25).Lysosomal trafficking of DOR, in contrast, is not prevented by mutation of cytoplasmic lysine residues (26) and can be regulated by ubiquitination-independent protein interaction(s) (27, 28). Nevertheless, both wild type and lysyl-mutant DORs traffic to lysosomes via a similar pathway as ubiquitin-dependent membrane cargo and require both HRS and active VPS4 to do so (29). These observations indicate that DOR engages the same core endocytic mechanism utilized by ubiquitination-directed membrane cargo but leave unresolved whether ubiquitination of DOR plays any role in this important cellular mechanism of receptor down-regulation.There is no doubt that DOR can undergo significant ubiquitination in mammalian cells, including HEK293 cells (30–32), where lysosomal trafficking of lysyl-mutant receptors was first observed (26). Ubiquitination was shown previously to promote proteolysis of DOR by proteasomes and to function in degrading misfolded receptors from the biosynthetic pathway (30, 31). A specific role of ubiquitination in promoting proteasome- but not lysosome-mediated proteolysis of DOR has been emphasized (32) and proposed to contribute to proteolytic down-regulation of receptors also from the plasma membrane (33).To our knowledge, no previous studies have determined if DOR ubiquitination plays any role in controlling receptor proteolysis mediated by lysosomes, although this represents a predominant pathway by which receptors undergo rapid down-regulation following ligand-induced endocytosis in a number of cell types, including HEK293 cells (8). In the present study, we have taken two approaches to addressing this fundamental question. First, we have investigated in greater detail the effects of lysyl-mutation on DOR ubiquitination and trafficking. Second, we have independently investigated the role of ubiquitination in controlling lysosomal proteolysis of wild type DOR. Our results clearly establish the ability of DOR to traffic efficiently to lysosomes in the absence of any detectable ubiquitination. Further, they identify a distinct and unanticipated function of AIP4-dependent ubiquitination in regulating the later proteolytic processing of receptors and show that this distinct ubiquitin-dependent regulatory mechanism operates effectively downstream of the sorting decision that commits internalized receptors for delivery to lysosomes.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Characterization of the ATP-dependent Sphingosine 1-Phosphate Transporter in Rat Erythrocytes

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5′-(β,γ-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent K_m value of 21 μm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.Sphingosine 1-phosphate (S1P),² a bioactive lipid molecule present in the blood, plays an important role in diverse cellular responses, such as migration, proliferation, and differentiation (1, 2). These processes are triggered by the binding of S1P to its specific receptors (3), of which five subtypes (S1P₁-S1P₅) have been identified in endothelial and immune cells (4). Studies using S1P₁ receptor-deficient mice showed abnormalities in lymphocyte egress from lymph nodes, spleen, and thymus (5, 6). Whereas blood plasma contains a basal level of S1P from the nanomolar to the micromolar range (7–12), lymphoid tissues maintain a low S1P environment through the activity of S1P lyase (13). It has been proposed that a higher concentration of S1P in the blood plasma than in the lymphoid organs establishes an essential gradient along which lymphocytes expressing the S1P₁ receptor on cell surfaces migrate (2, 5, 6, 13–15).The source of plasma S1P remains unclear despite its importance in the cellular responses of endothelial cells and lymphocytes. Unlike most cells, blood cells, astrocytes, and vascular endothelial cells are reported to release S1P (8, 16–18). These cells contain sphingosine kinase, which synthesizes S1P through the phosphorylation of sphingosine (16, 18, 19). Whereas platelets and mast cells release S1P in a stimulus-dependent manner (17, 20), erythrocytes, neutrophils, and mononuclear cells release S1P in a stimulus-independent manner (16). The roles of S1P derived from erythrocytes, the most abundant of these blood cells, have not been elucidated. However, recent reports suggest that S1P released from erythrocytes is a major source of plasma S1P (7, 9) and promotes lymphocyte egress to blood (9).Previously, we showed that S1P is released from rat platelets upon stimulation by thrombin or Ca²⁺ (21). We proposed that an ATP-dependent transporter plays a key role in S1P release from platelets (21). However, the detailed mechanism of S1P release is unclear because there is no way to assay the transport of S1P across the membrane. In this study we compared the properties of S1P release from erythrocytes with that of platelets and showed that S1P release from erythrocytes does not require any stimuli. We then established an assay to measure the ATP-dependent S1P uptake into inside-out membrane vesicles (IOVs) prepared from rat erythrocytes and characterized S1P transport in erythrocytes.     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Requirement of Myosin Vb��Rab11a��Rab11-FIP2 Complex in Cholesterol-regulated Translocation of NPC1L1 to the Cell Surface

Niemann-Pick C1-like 1 (NPC1L1) plays a critical role in the enterohepatic absorption of free cholesterol. Cellular cholesterol depletion induces the transport of NPC1L1 from the endocytic recycling compartment to the plasma membrane (PM), and cholesterol replenishment causes the internalization of NPC1L1 together with cholesterol via clathrin-mediated endocytosis. Although NPC1L1 has been characterized, the other proteins involved in cholesterol absorption and the endocytic recycling of NPC1L1 are largely unknown. Most of the vesicular trafficking events are dependent on the cytoskeleton and motor proteins. Here, we investigated the roles of the microfilament and microfilament-associated triple complex composed of myosin Vb, Rab11a, and Rab11-FIP2 in the transport of NPC1L1 from the endocytic recycling compartment to the PM. Interfering with the dynamics of the microfilament by pharmacological treatment delayed the transport of NPC1L1 to the cell surface. Meanwhile, inactivation of any component of the myosin Vb·Rab11a·Rab11-FIP2 triple complex inhibited the export of NPC1L1. Expression of the dominant-negative mutants of myosin Vb, Rab11a, or Rab11-FIP2 decreased the cellular cholesterol uptake by blocking the transport of NPC1L1 to the PM. These results suggest that the efficient transport of NPC1L1 to the PM is dependent on the microfilament-associated myosin Vb·Rab11a·Rab11-FIP2 triple complex.Cholesterol homeostasis in human bodies is maintained through regulated cholesterol synthesis, absorption, and excretion. Intestinal cholesterol absorption is one of the major pathways to maintain cholesterol balance. NPC1L1 (Niemann-Pick C1-like protein 1), a polytopic transmembrane protein highly expressed in the intestine and liver, is required for dietary cholesterol uptake and biliary cholesterol reabsorption (1–4). Genetic or pharmaceutical inactivation of NPC1L1 significantly inhibits cholesterol absorption and confers the resistance to diet-induced hypercholesterolemia (1, 2, 4). Ezetimibe, an NPC1L1-specific inhibitor, is currently used to prevent and treat cardiovascular diseases (5).Human NPC1L1 contains 1,332 residues with 13 transmembrane domains (6). The third to seventh transmembrane helices constitute a conserved sterol-sensing domain (4, 7). NPC1L1 recycles between the endocytic recycling compartment (ERC)³ and the plasma membrane (PM) in response to the changes of cholesterol level (8). ERC is a part of early endosomes that is involved in the recycling of many transmembrane proteins. It is also reported that ERC is a pool for free cholesterol storage (9). When cellular cholesterol concentration is low, NPC1L1 moves from the ERC to the PM (8, 10). Under cholesterol-replenishing conditions, NPC1L1 and cholesterol are internalized together and transported to the ERC (8). Disruption of microfilament, depletion of the clathrin·AP2 complex, or ezetimibe treatment can impede the endocytosis of NPC1L1, thereby decreasing cholesterol internalization (8, 10, 11).The microfilament (MF) system, part of the cytoskeleton network, is required for multiple cellular functions such as cell shape maintenance, cell motility, mitosis, protein secretion, and endocytosis (12, 13). The major players in the microfilament system are actin fibers and motor proteins (14). Actin fibers form a network that serves as the tracks for vesicular transport (15, 16). Meanwhile, the dynamic assembly and disassembly of actin fibers and the motor proteins provides the driving force for a multitude of membrane dynamics including endocytosis, exocytosis, and vesicular trafficking between compartments (15, 16).Myosins are a large family of motor proteins that are responsible for actin-based mobility (14). Class V myosins (17, 18), comprising myosin Va, Vb, and Vc, are involved in a wide range of vesicular trafficking events in different mammalian tissues. Myosin Va is expressed mainly in neuronal tissues (19, 20), whereas myosins Vb and Vc are universally expressed with enrichment in epithelial cells (21, 22). Class V myosins are recruited to their targeting vesicles by small GTPase proteins (Rab) (23). Rab11a and Rab11 family-interacting protein 2 (Rab11-FIP2) facilitate the binding of myosin Vb to the cargo proteins of endocytic recycling vesicles (24–28).Myosin Vb binds Rab11a and Rab11-FIP2 through the C-terminal tail (CT) domain. The triple complex of myosin Vb, Rab11a, and Rab11-FIP2 is critical for endocytic vesicular transport and the recycling of many proteins including transferrin receptor (29), AMPA receptors (30), CFTR (28), GLUT4 (31, 32), aquaporin-2 (26), and β2-adrenergic receptors (33). The myosin Vb-CT domain (24) competes for binding to Rab11a and Rab11-FIP2 and functions as a dominant-negative form. Expression of the CT domain substantially impairs the transport of vesicles. Deficient endocytic trafficking is also observed in cells expressing the GDP-locked form of Rab11a (S25N) (34) or a truncated Rab11-FIP2, which competes for the rab11a binding (35).Here we investigated the roles of actin fibers and motor proteins in the cholesterol-regulated endocytic recycling of NPC1L1. Using pharmaceutical inactivation, dominant-negative forms, and an siRNA technique, we demonstrated that actin fibers and myosin Vb·Rab11a·Rab11-FIP2 triple complex are involved in the export of NPC1L1 to the PM and that this intact MF-associated triple complex is required for efficient cholesterol uptake. Characterization of the molecules involved in the recycling of NPC1L1 may shed new light upon the mechanism of cholesterol absorption.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.