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The monomorphic antigen-presenting molecule MHC-I-related protein 1 (MR1) presents small molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and non-communicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included β2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knock-out of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key post-transcriptional determinant of MR1 surface expression.

The monomorphic antigen presenting molecule MHC-I-related protein 1 (MR1) presents small molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and non-communicable diseases and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialised antigen presentation pathway. Yet, the proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included β2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-ATPase in the endoplasmic reticulum (ER) with putative transporter function not previously associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knock-out of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 as a key post-transcriptional determinant of MR1 surface expression.

SUMMARYInsertion of hydrophobic transmembrane domains (TMDs) into the endoplasmic reticulum (ER) lipid bilayer is an essential step during eukaryotic membrane protein biogenesis. The ER membrane complex (EMC) functions as an insertase for TMDs of low hydrophobicity and is required for the biogenesis of a subset of tail-anchored (TA) and polytopic membrane proteins, including rhodopsin-1 (Rh1) and the TRP channel. To better understand the physiological implications of membrane protein biogenesis dependent on the EMC, we performed a bioinformatic analysis to predict TA proteins present in the Drosophila proteome. From 254 predicted TA proteins, subsequent genetic screening in Drosophila larval eye discs led to the identification of 2 proteins that require EMC for their biogenesis: farinelli (fan) and Xport-A. Fan is required for sperm individualization and male fertility in Drosophila and we now show that EMC is also required for these important biological processes. Interestingly, Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC loss of function mutations is secondary to the Xport-A defect. We show that EMC is required for Xport-A TMD membrane insertion and increasing the hydrophobicity of Xport-A TMD rendered its membrane insertion to become EMC-independent. Moreover, these EMC-independent Xport-A mutants rescued Rh1 and TRP biogenesis in EMC mutants. Our data establish that EMC can impact the biogenesis of polytopic membrane proteins indirectly, by controlling the biogenesis and membrane insertion of an essential protein co-factor.

Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 different stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.

Integral membrane proteins play key functional roles at organelles and the plasma membrane, necessitating their efficient and accurate biogenesis to ensure appropriate targeting and activity. The endoplasmic reticulum membrane protein complex (EMC) has recently emerged as an important eukaryotic complex for biogenesis of integral membrane proteins by promoting insertion and stability of atypical and sub-optimal transmembrane domains (TMDs). Although confirmed as a bona fide complex almost a decade ago, light is just now being shed on the mechanism and selectivity underlying the cellular responsibilities of the EMC. In this Review, we revisit the myriad of functions attributed the EMC through the lens of these new mechanistic insights, to address questions of the cellular and organismal roles the EMC has evolved to undertake., Summary: The EMC is an important factor facilitating membrane protein biogenesis. Here we discuss the broad cellular and organismal responsibilities overseen by client proteins requiring the EMC for maturation.

Significance While there is a clear understanding of how invariant NKT (iNKT) cells are activated in foreign infection, it remains unclear how they are activated during sterile inflammation, including cancer, where they have a well-defined role in tumor immunosurveillance. Here we elucidate a mechanism by which iNKT cells are activated through 1) the presentation of self-lipid antigens by endoplasmic reticulum-stressed antigen-presenting cells and 2) enhanced functional avidity driven by actin cytoskeletal remodeling. We further provide evidence that this mechanism of activation is at play in tumor settings. Here we describe a physiological context, relevant to human health and disease, that drives the presentation of immunogenic self-lipids to activate iNKT cells during sterile inflammation., Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.

How endoplasmic reticulum (ER) stress leads to cytotoxicity is ill-defined. Previously we showed that HeLa cells readjust homeostasis upon proteostatically driven ER stress, triggered by inducible bulk expression of secretory immunoglobulin M heavy chain (μs) thanks to the unfolded protein response (UPR; Bakunts et al., 2017). Here we show that conditions that prevent that an excess of the ER resident chaperone (and UPR target gene) BiP over µs is restored lead to µs-driven proteotoxicity, i.e. abrogation of HRD1-mediated ER-associated degradation (ERAD), or of the UPR, in particular the ATF6α branch. Such conditions are tolerated instead upon removal of the BiP-sequestering first constant domain (CH1) from µs. Thus, our data define proteostatic ER stress to be a specific consequence of inadequate BiP availability, which both the UPR and ERAD redeem.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

The eukaryotic endoplasmic reticulum (ER) membrane contains essential complexes that oversee protein biogenesis and lipid metabolism, impacting nearly all aspects of cell physiology. The ER membrane protein complex (EMC) is a newly described transmembrane domain (TMD) insertase linked with various phenotypes, but whose clients and cellular responsibilities remain incompletely understood. We report that EMC deficiency limits the cellular boundaries defining cholesterol tolerance, reflected by diminished viability with limiting or excessive extracellular cholesterol. Lipidomic and proteomic analyses revealed defective biogenesis and concomitant loss of the TMD-containing ER-resident enzymes sterol-O-acyltransferase 1 (SOAT1) and squalene synthase (SQS, also known as FDFT1), which serve strategic roles in the adaptation of cells to changes in cholesterol availability. Insertion of the weakly hydrophobic tail-anchor (TA) of SQS into the ER membrane by the EMC ensures sufficient flux through the sterol biosynthetic pathway while biogenesis of polytopic SOAT1 promoted by the EMC provides cells with the ability to store free cholesterol as inert cholesteryl esters. By facilitating insertion of TMDs that permit essential mammalian sterol-regulating enzymes to mature accurately, the EMC is an important biogenic determinant of cellular robustness to fluctuations in cholesterol availability. This article has an associated First Person interview with the first author of the paper., Highlighted Article: The ER membrane protein complex promotes biogenesis of key membrane-bound enzymes responsible for regulation of cholesterol biosynthesis and storage, an important determinant of mammalian cell viability.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn

Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β‐catenin by the ubiquitin‐proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)‐associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP‐dependent manner. Ubiquitination of Evi involves the E2‐conjugating enzyme UBE2J2 and the E3‐ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD‐dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.

The mammalian ubiquitin ligase Hrd1 is the central component of a complex facilitating degradation of misfolded proteins during the ubiquitin–proteasome-dependent process of ER-associated degradation (ERAD). Hrd1 associates with cofactors to execute ERAD, but their roles and how they assemble with Hrd1 are not well understood. Here, we identify crucial cofactor interaction domains within Hrd1 and report a previously unrecognised evolutionarily conserved segment within the intrinsically disordered cytoplasmic domain of Hrd1 (termed the HAF-H domain), which engages complementary segments in the cofactors FAM8A1 and Herp (also known as HERPUD1). This domain is required by Hrd1 to interact with both FAM8A1 and Herp, as well as to assemble higher-order Hrd1 complexes. FAM8A1 enhances binding of Herp to Hrd1, an interaction that is required for ERAD. Our findings support a model of Hrd1 complex formation, where the Hrd1 cytoplasmic domain and FAM8A1 have a central role in the assembly and activity of this ERAD machinery.

The four muscle-type nicotinic acetylcholine receptor (AChR) subunits, alpha, beta, gamma, and delta, assemble into functional alpha(2)betagammadelta pentamers in the endoplasmic reticulum (ER) through a series of interdependent folding and oligomerization events. The first stable assembly intermediate is a trimer composed of alpha, beta, and gamma subunits. The formation of alphabetagamma trimers initiates a series of subunit folding and processing events that allow addition of delta subunits to form alphabetagammadelta tetramers. Subunit folding and processing continue with formation of the ligand-binding sites on the alpha subunit of alphabetagammadelta tetramers and the second alpha subunit added to assemble alpha(2)betagammadelta pentamers. AChR assembly is inefficient. Only 20-30% of synthesized subunits assemble into mature receptors in the ER, while the remaining unassembled subunits are degraded. However, the efficiency of subunit assembly can be regulated under certain conditions leading to higher AChR expression. Increased intracellular cAMP levels cause a 2- to 3-fold increase in AChR assembly efficiency and a comparable increase in surface expression. Additionally, block of ubiquitin-proteasome degradation appears to enhance AChR assembly and expression. Thus, the regulation of AChR assembly through posttranslational mechanisms is a potential therapeutic target for increasing AChR expression in diseases in which expression is compromised.

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. While it is clear that IP(3) receptors are polyubiquitinated and are transferred to the proteasome by a p97-based complex, currently very little is known about the proteins that initially select activated IP(3) receptors for ERAD. Here, we have transfected HeLa cells to stably express m3 muscarinic receptors to allow for the study of IP(3) receptor ERAD in this cell type, and show that IP(3) receptors are polyubiquitinated and then degraded by the proteasome in response to carbachol, a muscarinic agonist. In seeking to identify proteins that mediate IP(3) receptor ERAD we found that both SPFH1 and SPFH2 (also known as erlin 1 and erlin 2), which exist as a hetero-oligomeric complex, rapidly associate with IP(3) receptors in a manner that precedes polyubiquitination and the association of p97. Suppression of SPFH1 and SPFH2 expression by RNA interference markedly inhibited carbachol-induced IP(3) receptor polyubiquitination and degradation, but did not affect carbachol-induced calcium mobilization or IkappaBalpha processing, indicating that the SPFH1/2 complex is a key player in IP(3) receptor ERAD, acting at a step after IP(3) receptor activation, but prior to IP(3) receptor polyubiquitination. Suppression of SPFH1 and SPFH2 expression had only slight effects on the turnover of some exogenous model ERAD substrates, and had no effect on sterol-induced ERAD of endogenous 3-hydroxy-3-methylglutaryl-CoA reductase. Overall, these studies show that m3 receptor-expressing HeLa cells are a valuable system for studying IP(3) receptor ERAD, and suggest that the SPFH1/2 complex is a factor that selectively mediates the ERAD of activated IP(3) receptors.

CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous aggregates in mouse models of polyglutamine disease, but the cellular mechanism underlying this phenomenon is unknown. Here, we show that autophagy is essential for the elimination of aggregated forms of mutant huntingtin and ataxin-1 from the cytoplasmic but not nuclear compartments. Human orthologs of yeast autophagy genes, molecular determinants of autophagic vacuole formation, are recruited to cytoplasmic but not nuclear inclusion bodies in vitro and in vivo . These data indicate that autophagy is a critical component of the cellular clearance of toxic protein aggregates and may help to explain why protein aggregates are more toxic when directed to the nucleus.

© 2015 Elsevier Inc. Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating thedegradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.

Misfolded proteins of the endoplasmic reticulum (ER) are eliminated by the ER-associated degradation (ERAD) in eukaryotes. In S. cerevisiae, ER-resident lectins mediate substrate recognition through bipartite signals consisting of an unfolded local structure and the adjacent glycan. Trimming of the glycan is essential for the directional delivery of the substrates. Whether a similar recognition and delivery mechanism exists in mammalian cells is unknown. In this study, we systematically study the function and substrate specificity of known mammalian ER lectins, including EDEM1/2/3, OS-9 and XTP-3B using the recently identified ERAD substrate sonic hedgehog (SHH), a soluble protein carrying a single N-glycan, as well as its nonglycosylated mutant N278A. Efficient ERAD of N278A requires the core processing complex of HRD1, SEL1L and p97, similar to the glycosylated SHH. While EDEM2 was required for ERAD of both glycosylated and non-glycosylated SHHs, EDEM3 was only necessary for glycosylated SHH and EDEM1 was dispensable for both. Degradation of SHH and N278A also required OS-9, but not the related lectin XTP3-B. Robust interaction of both EDEM2 and OS-9 with a non-glycosylated SHH variant indicates that the misfolded polypeptide backbone, rather than a glycan signature, functions as the predominant signal for recognition for ERAD. Notably, SHH-N278A is the first nonglycosylated substrate to require EDEM2 for recognition and targeting for ERAD. EDEM2 also interacts with calnexin and SEL1L, suggesting a potential avenue by which misfolded glycoproteins may be shunted towards SEL1L and ERAD rather than being released into the secretory pathway. Thus, ER lectins participate in the recognition and delivery of misfolded ER substrates differently in mammals, with an underlying mechanism distinct from that of S. cerevisiae.

The endoplasmic reticulum–associated degradation (ERAD) pathway maintains protein homeostasis in the ER by retrotranslocating unwanted proteins to the cytosol for proteasomal degradation. This Review discusses the integral role of the ubiquitin system in ERAD, highlighting how the two pathways intertwine to facilitate transport across the ER membrane. The eukaryotic endoplasmic reticulum (ER) maintains protein homeostasis by eliminating unwanted proteins through the evolutionarily conserved ER-associated degradation (ERAD) pathway. During ERAD, maturation-defective and surplus polypeptides are evicted from the ER lumen and/or lipid bilayer through the process of retrotranslocation and ultimately degraded by the proteasome. An integral facet of the ERAD mechanism is the ubiquitin system, composed of the ubiquitin modifier and the factors for assembling, processing and binding ubiquitin chains on conjugated substrates. Beyond simply marking polypeptides for degradation, the ubiquitin system is functionally intertwined with retrotranslocation machinery to transport polypeptides across the ER membrane.

The Cancer/Testis (CT) antigen family of genes are transcriptionally repressed in most human tissues but are atypically re-expressed in many malignant tumour types. Their restricted expression profile makes CT antigens ideal targets for cancer immunotherapy. As little is known about whether CT antigens may be regulated by post-translational processing, we investigated the mechanisms governing degradation of NY-ESO-1 and MAGE-C1 in selected cancer cell lines. Inhibitors of proteasome-mediated degradation induced the partitioning of NY-ESO-1 and MAGE-C1 into a detergent insoluble fraction. Moreover, this treatment also resulted in increased localisation of NY-ESO-1 and MAGE-C1 at the centrosome. Despite their interaction, relocation of either NY-ESO-1 or MAGE-C1 to the centrosome could occur independently of each other. Using a series of truncated fragments, the regions corresponding to NY-ESO-1(91-150) and MAGE-C1(900-1116) were established as important for controlling both stability and localisation of these CT antigens. Our findings demonstrate that the steady state levels of NY-ESO-1 and MAGE-C1 are regulated by proteasomal degradation and that both behave as aggregation-prone proteins upon accumulation. With proteasome inhibitors being increasingly used as front-line treatment in cancer, these data raise issues about CT antigen processing for antigenic presentation and therefore immunogenicity in cancer patients.

The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system. ERAD proceeds through four tightly coupled steps involving substrate selection, dislocation across the ER membrane, covalent conjugation with polyubiquitin, and proteasomal degradation. The ERAD machinery shows a modular organization with central ER membrane-embedded ubiquitin ligases linking components responsible for recognition in the ER lumen to the ubiquitin-proteasome system in the cytoplasm. The core ERAD machinery is highly conserved among eukaryotes and much of our basic understanding of ERAD organization has been derived from genetic and biochemical studies of yeast. In this article we discuss how the core ERAD machinery is organized in mammalian cells.

Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.

The AAA-ATPase p97/VCP facilitates protein dislocation during endoplasmic reticulum-associated degradation (ERAD). To understand how p97/VCP accomplishes dislocation, a series of point mutants was made to disrupt distinguishing structural features of its central pore. Mutants were evaluated in vitro for ATPase activity in the presence and absence of synaptotagmin I (SytI) and in vivo for ability to process the ERAD substrate TCRalpha. Synaptotagmin induces a 4-fold increase in the ATPase activity of wild-type p97/VCP (p97/VCP(wt)), but not in mutants that showed an ERAD impairment. Mass spectrometry of crosslinked synaptotagmin . p97/VCP revealed interactions near Trp551 and Phe552. Additionally, His317, Arg586, and Arg599 were found to be essential for substrate interaction and ERAD. Except His317, which serves as an interaction nexus, these residues all lie on prominent loops within the D2 pore. These data support a model of substrate dislocation facilitated by interactions with p97/VCP's D2 pore.

Control of ligand-gated ion channel (LGIC) expression is essential for the formation, maintenance and plasticity of synapses. Treatment of mouse myotubes with proteasome inhibitors increased the number of surface nicotinic acetylcholine receptors (AChRs), indicating LGIC expression is regulated by the ubiquitin-proteasome system (UPS). Elevated surface expression resulted from increased AChR delivery to the plasma membrane and not from decreased turnover from the surface. The rise in AChR trafficking was the direct result of increased assembly of subunits in the endoplasmic reticulum (ER). Because proteasome inhibitors also blocked ER-associated degradation (ERAD) of unassembled AChR subunits, the data indicate that the additional AChRs were assembled from subunits normally targeted for ERAD. Our data show that AChR surface expression is regulated by the UPS through ERAD, whose activity determines oligomeric receptor assembly efficiency.

A case of primary cutaneous cryptococcal infection is presented and cases of primary cutaneous cryptococcosis in normal and immunocompromised hosts are reviewed. Cutaneous cryptococcosis can occur from local inoculation or dissemination from a distant site of infection. Risk factors associated with development of primary cutaneous cryptococcosis are those which affect cell-mediated immunity, such as corticosteroid usage, solid organ transplantation, sarcoidosis and immunosuppression. The cutaneous manifestations of cryptococcosis are protean and may mimic other cutaneous diseases. Patients with a diagnosis of cryptococcosis from a skin biopsy or culture should undergo evaluation to exclude disseminated disease and an evaluation of cell-mediated immunity. Although some patients do well without antifungal therapy, these patients cannot be discerned prospectively and therefore antifungal therapy appears warranted in all patients with localized disease. Choice of therapy depends on the extent of disease and immunocompetence of the host.

Presenilin 1 (PS1) and presenilin 2 play a critical role in the gamma-secretase processing of amyloid precursor protein (APP) and Notch1. Here, we investigate maturation and intracellular trafficking of APP and other membrane proteins in cells expressing an experimental PS1 deletion mutant (deltaM1,2). Stable expression of deltaM1,2 impairs gamma-secretase processing of Notch1 and delays Abeta secretion. Kinetic studies show enhanced O-glycosylation and sialylation of holo-APP and marked accumulation of APP COOH-terminal fragments (CTFs). Surface biotinylation, live staining, and trafficking studies show increased surface accumulation of holo-APP and CTFs in deltaM1,2 cells resulting from enhanced surface delivery of newly synthesized APP. Expression of a loss-of-function PS1 mutant (D385A) or incubation of cells with gamma-secretase inhibitors also increases surface levels of holo-APP and CTFs. In contrast to APP, glycosylation and surface accumulation of another type I membrane protein, nicastrin, are markedly reduced in deltaM1,2 cells. Finally, expression of deltaM1,2 results in the increased assembly and surface expression of nicotinic acetylcholine receptors, illustrating that PS1's influence on protein trafficking extends beyond APP and other type I membrane protein substrates of gamma-secretase. Collectively, our findings provide evidence that PS1 regulates the glycosylation and intracellular trafficking of APP and select membrane proteins.

Frequency (mean and median power frequency, f and fm) and amplitude (average rectified and root mean square values, ARV and rms), parameters of the M-wave, and the dorsiflexor force parameters of the anterior tibial muscles were measured in seven healthy human subjects. Intermittent, voluntary contractions at relative intensities (40%, 60%, and 80%) of maximal voluntary contraction (MVC) were performed in conjunction with electrical stimulation. The M-wave parameter changes were measured over the course of the isometric contractions. At higher force levels, M-wave potentiation was observed as increases in both ARV and rms. The ARV augmentation attained levels as high as 206.1 (SD 7.4)% of resting values after both initial and final contractions of 80% MVC, reaching statistical significance (P < 0.01). The f and fm failed to show a significant difference at any level of contraction. It was surmised that potentiation of the M-wave was the result of an increased contribution of muscle fibre type IIb recruited during higher contraction levels, reflecting the change to larger, deeper innervating motoneurons as the intensity of contraction, as a percentage of MVC, rose. Recruitment of type IIb fibres, which have been reported to have a higher energy potential and frequency content, were thought to reflect changes in the local excitability threshold of some motor units as the force intensity increased during the intermittent voluntary contractions. It is suggested that the M-wave elicited after contractions has the potential to reflect, to some extent, motor unit recruitment changes resulting from the preceding contractions, and that through comparisons of M-wave amplitude parameters contributions of varying fibre types over the course of a contraction may be indicated.

A biological treatment process employing immobilized microbial populations was field tested on contaminated ground water having dilute concentrations of volatile organics and organic pesticides. The process, consisting of two packed bed reactors in series containing specific adapted microbial strains immobilized on a porous diatomaceous earth support, was operated in a plug flow configuration over a 90-day period at a flow rate of 80 gallons per day (gpd). Process viability of this multiple microbe/multiple toxicant treatment approach was determined based upon comparisons to carbon adsorption technology.