ClC-2 is a broadly distributed chloride channel with an enigmatic neurophysiological function. In this issue of Neuron, Jeworutzki et al. (2012) use a biochemical approach to identify GlialCAM, a protein with a defined link to leukodystrophy, as a ClC-2 auxiliary subunit.

Numerous physiological functions, including a role as a cell surface receptor, have been ascribed to Alzheimer's disease-associated amyloid precursor protein (APP). However, detailed analysis of intracellular signaling mediated by APP in neurons has been lacking. Here, we characterized intrinsic signaling associated with membrane-bound APP C-terminal fragments, which are generated following ...

Bacteriorhodopsin (BR) mutagenesis plays an important role in the development of BR-based materials and tools with enhanced optical and electrical properties. Previously reported protocols for generating BR mutations are inefficient for the preparation and purification of mutant proteins. Therefore, a series of BR mutations were generated by using improved methods, ...

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis ...

Virions of the Human Immunodeficiency Virus (HIV) infect cells by first attaching with their surface spikes to the CD4 receptor on target cells. This leads to conformational changes in the viral spikes, enabling the virus to engage a coreceptor, commonly CCR5 or CXCR4, and consecutively to insert the fusion peptide ...

Eps15 homology domain (EHD) proteins are conserved adenosine triphosphatases that are involved in membrane remodeling. EHD family members are structurally similar to the guanosine triphosphatase (GTPase) dynamin, and both are essential for the fission step of clathrin-mediated endocytosis. This Journal Club highlights a recent study by Jakobsson et al. that ...

SecA is a translocation ATPase that drives protein translocation. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it. This mutant was inactive as to proOmpA translocation, however, it generated a translocation intermediate of 18 kDa. Further addition of wild-type SecA caused its translocation into either mature ...

Sparsomycin is an antibiotic that targets the peptidyl transferase center of the ribosome and has the ability to promote ribosomal translocation in the absence of EF-G and GTP. Here we show that changes in the configurations at the two chiral centers of sparsomycin, especially at the chiral carbon, can greatly ...

SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high-affinity sites and at phospholipid low-affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the ...

During protein synthesis, mRNA and tRNAs are iteratively translocated by the ribosome. Precisely what molecular event is rate limiting for translocation is not known. Here we show that disruption of the interactions between the A-site codon and the ribosome accelerates translocation, suggesting that the release of the mRNA from the ...

Mitochondrial protein import requires cooperation of the machineries called translocators in the outer and inner mitochondrial membranes. Here we analyze the interactions of Tom22, a multifunctional subunit of the outer membrane translocator TOM40 complex, with other translocator subunits such as Tom20, Tom40, and Tim50 and with substrate precursor proteins at ...

Voorhees et al. (Reports, 5 November 2010, p. 835) determined the structure of elongation factor Tu (EF-Tu) and aminoacyl-transfer RNA bound to the ribosome with a guanosine triphosphate (GTP) analog. However, their identification of histidine-84 of EF-Tu as deprotonating the catalytic water molecule is problematic in relation to their atomic ...

Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of ...

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its ...

INT6/EIF3E has been implicated in breast tumorigenesis, but its functional activities remain poorly defined. We found that, repressing INT6 expression induced transformed properties in normal human mammary epithelium (MCF10A); in contrast, Int6 silencing induced apoptosis in HeLa cells. As in fission yeast, Int6 in human cells was required for assembly ...

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute ...

Disease-causing mutations of genes encoding small MW heat shock proteins (sHSPs) constitute a growing family of inherited myofibrillar disorders. In the present work, we found that three structurally-distinct CryAB mutants R120G, 450delA and 464delCT are mostly present in the detergent-insoluble fractions when overexpressed in H9c2 rat heart cells. We found ...

The MALT1 paracaspase has arginine-directed proteolytic activity. A20 is a dual ubiquitin-editing enzyme involved in termination of NF-κB signaling. Upon T- or B-cell receptor engagement human (h) A20 is cleaved by MALT1 after arginine 439, yielding an N-terminal fragment (hA20p50) and a C-terminal one (hA20p37). The hA20p50 fragment has never ...

The molecular details of the biogenesis of double-membraned autophagosomes are poorly understood. We identify the Saccharomyces cerevisiae AAA-adenosine triphosphatase Cdc48 and its substrate-recruiting cofactor Shp1/Ubx1 as novel components needed for autophagosome biogenesis. In mammals, the Cdc48 homologue p97/VCP and the Shp1 homologue p47 mediate Golgi reassembly by extracting an unknown ...

Expansion of a polyglutamine tract in ataxin-3 (polyQ) causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation. Several lines of evidence demonstrate that polyQ also accumulates in mitochondria and causes mitochondrial dysfunction. To uncover the mechanism of mitochondrial quality-control via the ubiquitin-proteasome pathway, we investigated whether MITOL, ...

Messenger RNA lacking stop codons ('non-stop mRNA') can arise from errors in gene expression, and encode aberrant proteins whose accumulation could be deleterious to cellular function. In bacteria, these 'non-stop proteins' become co-translationally tagged with a peptide encoded by ssrA/tmRNA (transfer-messenger RNA), which signals their degradation by energy-dependent proteases. How ...

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in ...

Post-translational modification of proteins with prokaryotic ubiquitin-like protein (Pup) is the bacterial equivalent of ubiquitination in eukaryotes. Mycobacterial pupylation is a two-step process in which the carboxy-terminal glutamine of Pup is first deamidated by Dop (deamidase of Pup) before ligation of the generated γ-carboxylate to substrate lysines by the Pup ...

The Cullin3-based E3 ubiquitin ligase complex is thought to play an important role in the cellular response to oxidative stress and xenobiotic assault. While limited biochemical studies of the ligase's role in these complex signaling pathways are beginning to emerge, structural studies are lagging far behind due to the inability ...

BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate ...

Mammalian alpha4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. alpha4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using alpha4 as bait ...

BRCC36 is a member of the JAMM/MPN(+) family of zinc metalloproteases that specifically cleaves Lys 63-linked polyubiquitin chains in vitro. We and others showed previously that BRCC36 is a component of the BRCA1-A complex, which consists of RAP80, CCDC98/ABRAXAS, BRCC45/BRE, MERIT40/NBA1, BRCC36, and BRCA1. This complex participates in the regulation ...

The small ubiquitin-related modifiers (SUMOs) are evolutionarily conserved polypeptides that are covalently conjugated to protein targets to modulate their subcellular localization, half-life, or activity. Steady-state SUMO conjugation levels increase in response to many different types of environmental stresses, but how the SUMO system is regulated in response to these insults ...

The characteristic increase in protein catabolism during muscle atrophy is largely the result of an increase in E3 ubiquitin ligase expression, specifically that of atrogin-1, or FBXO32, which functions to polyubiquitinate proteins. In rainbow trout, the cDNA sequences of two E3 ubiquitin ligase F-box proteins, FBXO32 and FBXO25, were characterized ...

Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the ...

Ubiquitin is a small polypeptide and ubiquitination is the post-translational modification by ubiquitin protein, resulting in degradation of target proteins by the 26S proteasome complex. Here, we found that E3 ubiquitin ligase SINAT5, an Arabidopsis homologue of the Drosophila SINA RING-finger protein, interacts directly with LHY, a component of the ...

At least four paralogs of the small ubiquitin-related modifier (SUMO) exist in humans, but there is limited information about SUMO paralogs from other vertebrate species. We isolated the four cDNA encoding proteins, similar to human SUMOs, from the medaka fish, Oryzias latipes: OlSUMO-1, OlSUMO-2, OlSUMO-3, and OlSUMO-4. The amino acid ...

Dynamic modification of target proteins by small ubiquitin-like modifier (SUMO) is known to modulate many important cellular processes and is required for cell viability and development in all eukaryotes. However, understanding of SUMO systems in plants, especially in unicellular green algae, remains elusive. In this study, Chlamydomonas reinhardtii CrSUMO96, CrSUMO97 ...

Protein sumoylation plays an important but poorly understood role in controlling genome integrity. In Saccharomyces cerevisiae, the Slx5-Slx8 SUMO-targeted Ub ligase appears to be needed to ubiquitinate sumoylated proteins that arise in the absence of the Sgs1 DNA helicase. WSS1, a high-copy-number suppressor of a mutant SUMO, was implicated in ...

We previously reported the identification of DP-1 isoforms (alpha and beta), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for ...

Translesion synthesis is a major mechanism with which eukaryotic cells deal with DNA damage during replication. Mono-ubiquitinated PCNA is a key regulator of this process. We have investigated whether a ubiquitin-PCNA fusion can mimic ubiquitinated PCNA, by transforming plasmids expressing this fusion protein into different mutants of Schizosaccharomyces pombe. We ...

A novel splice variant of hPirh2, named hPirh2b, was isolated from human fetal liver cDNA library. hPirh2b has a 38-nucleotide deletion and encodes a 188-amino acid protein with a truncated RING-H2 domain. It shows no ubiquitin protein ligase activity. A low level of expression of hPirh2 was found both at ...

Small ubiquitin-like modifier (SUMO) is a post-translational modifier peptide that is involved in several biological processes in eukaryotes. Arabidopsis SIZ1, a SUMO E3 ligase, is an ortholog of the mammalian PIAS (Protein Inhibitor of Activated STAT) and yeast SIZ (SAP/Miz) proteins. SIZ1 contains all of the typical domains of PIAS/SIZ-type ...

A prokaryotic protein tagging system called pupylation that is analogous to ubiquitylation in eukaryotes has recently been described. In this process, prokaryotic ubiquitin-like protein (Pup) is coupled to substrate proteins via an isopeptide bond in order to target them for degradation by the proteasome. The ligation occurs via a condensation ...

The valosin-containing protein (p97) is a ubiquitin-dependent ATPase that plays central roles in ubiquitin proteasome system (UPS)-mediated protein degradation pathways. p97 has been recently identified as a putative substrate of active Caspase-6 (Casp6) in primary human neurons. Since Casp6 is activated in mild cognitive impairment (MCI) and Alzheimer's disease (AD) ...

RIG-I detects invading viral RNA and activates the transcription factors NF-kappaB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5'-triphosphate strongly activates the RIG-I-IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria, and cytosol. Activation of RIG-I requires not only RNA but also ...

Varicella zoster virus encodes an immediate-early (IE) protein termed ORF61p that is orthologous to the herpes simplex virus IE protein ICP0. Although these proteins share several functional properties, ORF61p does not fully substitute for ICP0. The greatest region of similarity between these proteins is a RING finger domain. We demonstrate ...

Cupriavidus necator JMP134 utilizes meta-nitrophenol (MNP) as the sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degradation performed in silico suggested that MnpC might have played an important role in MNP degradation. In order to experimentally confirm the prediction, we have now characterized the mnpC-encoded (amino)hydroquinone ...

Yeast permeases, that act as transporters for nutrients including amino acids, nucleobases and metals, provide a powerful model system for dissecting the physiological control of membrane protein trafficking. Modification of these transporters by ubiquitin is known to target them for degradation in the vacuole, the degradation organelle of fungi. Recent ...

Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and ...

The N-end rule pathway is a highly conserved process that operates in many different organisms. It relates the metabolic stability of a protein to its N-terminal amino acid. Consequently, amino acids are described as either 'stabilizing' or 'destabilizing'. Destabilizing residues are organized into three hierarchical levels: primary, secondary, and in ...

An expanded polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt) causes misfolding and accumulation of htt in neuronal cells and the subsequent neurodegeneration of Huntington's disease (HD). Clearing the misfolded htt is critical for preventing neuropathology, and this process is mediated primarily by both the ubiquitin-proteasome system (UPS) ...

MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21(Waf1). The present study was designed to characterize the mechanism(s) by which MDM2 induces p21(Waf1) degradation. We first determined the ...

In addition to threonine auxotrophy, mutation of the Saccharomyces cerevisiae threonine biosynthetic genes THR1 (encoding homoserine kinase) and THR4 (encoding threonine synthase) results in a plethora of other phenotypes. We investigated the basis for these other phenotypes and found that they are dependent on the toxic biosynthetic intermediate homoserine. Moreover, ...

Serotonin N-acetyltransferase (AANAT) catalyzes the conversion of serotonin to N-acetylserotonin, which is the immediate precursor for formation of melatonin. Although it is known that AANAT is degraded via the proteasomal proteolysis, detailed mechanisms are not defined. In this paper, we tested the in vivo role of proteasome inhibition on AANAT ...