Biomedical subjects

James E Rothman

Publications and source records attributed to James E Rothman.

At least 19 recordsLinked to original sources

PubMed2007

Selective activation of cognate SNAREpins by Sec1/Munc18 proteins.

Sec1/Munc18 (SM) proteins are required for every step of intracellular membrane fusion, but their molecular mechanism of action has been unclear. In this work, we demonstrate a fundamental role of the SM protein: to act as a stimulatory subunit of its cognate SNARE fusion machinery. In a reconstituted system, mammalian SNARE pairs assemble between bilayers to drive a basal fusion reaction. Munc18-1/nSec1, a synaptic SM protein required for neurotransmitter release, strongly accelerates this reaction through direct contact with both t- and v-SNAREs. Munc18-1 accelerates fusion only for the cognate SNAREs for exocytosis, therefore enhancing fusion specificity.

Amino Acid Motifs↗

PubMed2006

Lipidic antagonists to SNARE-mediated fusion.

SNARE proteins mediate the fusion of lipid bilayers by the directed assembly of coiled-coil domains arising from apposing membranes. We have utilized inverted cone-shaped lipids, antagonists of the necessary membrane deformation during fusion to characterize the extent and range of SNARE assembly up to the moment of stalk formation between bilayers. The inverted cone-shaped lipid family of acyl-CoAs specifically inhibits the completion of fusion in an acyl-chain length-dependent manner. Removal of acyl-CoA from the membrane relieves the inhibition and initiates a burst of membrane fusion with rates exceeding any point in the control curves lacking acyl-CoA. This burst indicates the accumulation of semi-assembled fusion complexes. These preformed complexes are resistant to cleavage by botulinum toxin B and thus appear to have progressed beyond the "loosely zippered" state of docked synaptic vesicles. Surprisingly, application of the soluble domain of VAMP2, which blocks SNARE assembly by competing for binding on the available t-SNAREs, blocks recovery from the acyl-CoA inhibition. Thus, complexes formed in the presence of a lipidic antagonist to fusion are incompletely assembled, suggesting that the formation of tightly assembled SNARE pairs requires progression all the way through to membrane fusion. In this regard, physiologically docked exocytic vesicles may be anchored by a highly dynamic and potentially even reversible SNAREpin.

Acyl Coenzyme A↗

PubMed2006

A clamping mechanism involved in SNARE-dependent exocytosis.

During neurotransmitter release at the synapse, influx of calcium ions stimulates the release of neurotransmitter. However, the mechanism by which synaptic vesicle fusion is coupled to calcium has been unclear, despite the identification of both the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal calcium sensor (synaptotagmin). Here, we describe what may represent a basic principle of the coupling mechanism: a reversible clamping protein (complexin) that can freeze the SNAREpin, an assembled fusion-competent intermediate en route to fusion. When calcium binds to the calcium sensor synaptotagmin, the clamp would then be released. SNARE proteins, and key regulators like synaptotagmin and complexin, can be ectopically expressed on the cell surface. Cells expressing such "flipped" synaptic SNAREs fuse constitutively, but when we coexpressed complexin, fusion was blocked. Adding back calcium triggered fusion from this intermediate in the presence of synaptotagmin.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Functional mapping of disease susceptibility loci using cell biology.

In most genome-wide linkage studies, implication of a causative disease gene often requires years of expanding the study to more families and finer mapping of the initially described region. Even after such efforts, unobtainable sample sizes can be required to make statistically meaningful conclusions about a single gene. Here we demonstrate that by adding a layer of functional biology to statistical genetic results, this process can be accelerated. The diabetes susceptibility locus (chromosome 18p11) was systematically dissected by using a cell-based secretion assay and RNA interference, and we identified laminin alpha1 to have a role in pancreatic beta cell secretion. The screen was extended to identify laminin receptor 1 as a functional partner in regards to beta cell function. Our approach can potentially be widely used in the setting of high-throughput cellular screening of other loci to identify candidate genes.

Cell Line↗

PubMed2006

Autophagy-mediated clearance of huntingtin aggregates triggered by the insulin-signaling pathway.

Conditional mouse models of polyglutamine diseases, such as Huntington's disease (HD), have revealed that cells can clear accumulated pathogenic proteins if the continuous production of the mutant transgene is halted. Invariably, the clearance of the protein leads to regression of the disease symptoms in mice. In light of these findings, it is critical to determine the pathway responsible for alleviating this protein accumulation to define targets to fight these diseases. In a functional genetic screen of HD, we found that activation of insulin receptor substrate-2, which mediates the signaling cascades of insulin and insulin-like growth factor 1, leads to a macroautophagy-mediated clearance of the accumulated proteins. The macroautophagy is triggered despite activation of Akt, mammalian target of rapamycin (mTOR), and S6 kinase, but still requires proteins previously implicated in macroautophagy, such as Beclin1 and hVps34. These findings indicate that the accumulation of mutant protein can lead to mTOR-independent macroautophagy and that lysosome-mediated degradation of accumulated protein differs from degradation under conditions of starvation.

Animals↗

PubMed2006

Cell-based assays using primary endothelial cells to study multiple steps in inflammation.

Cell-based assays are powerful tools for drug discovery and provide insight into complex signal transduction pathways in higher eukaryotic cells. Information gleaned from assays that monitor a cellular phenotype can be used to elucidate the details of a single pathway and to establish patterns of cross talk between pathways. By selecting the appropriate cell model, cell-based assays can be used to understand the function of a specific cell type in a complex disease process such as inflammation. We have used human umbilical vein endothelial cells to establish three cell-based, phenotypic assays that query different stages of a major signaling pathway activated in inflammation. One assay analyzes the tumor necrosis factor alpha (TNFalpha)-induced translocation of the transcription factor NF-kappaB from the cytoplasm into the nucleus 20 min after stimulation with TNFalpha. Two more assays monitor the expression of E-selectin and VCAM-1, 4 and 24 h after stimulation with TNFalpha. Indirect immunofluorescence and high-throughput automated microscopy were used to analyze cells. Imaging was performed with the IN Cell Analyzer 3000. All assays proved to be highly robust. Z' values between 0.7 and 0.8 make each of the three assays well suited for use in high-throughput screening for drug or probe discovery.

Animals↗

PubMed2005

Dynamic transport of SNARE proteins in the Golgi apparatus.

Localization of a membrane protein in a subcellular compartment can be achieved by its retention in the compartment or by its continuous transport toward this compartment. Previous results have suggested that specific enzymes are localized in the Golgi apparatus at least in part by selective retention and exclusion from transport vesicles. However, the function of some Golgi SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins is not compatible with their exclusion from transport vesicles. To help understand the mechanism accounting for the localization of SNARE proteins in the Golgi apparatus, we analyzed their lateral distribution in the Golgi cisternae and their incorporation into transport vesicles. According to our results, all SNARE proteins are efficiently incorporated into transport vesicles, indicating that the localization of SNARE proteins in the Golgi apparatus is not based on a static retention mechanism. Detailed analysis suggested that incorporation into transport vesicles was more efficient for SNARE proteins restricted to the cis face of the Golgi as compared with SNAREs present at the trans face. Furthermore, overexpression of a cis-Golgi SNARE protein altered concomitantly its incorporation in transport vesicles and its intra-Golgi localization. These observations suggest that, contrary to resident Golgi enzymes, SNARE proteins are localized in the Golgi apparatus as the result of a dynamic transport equilibrium.

Animals↗

PubMed2005

SNAREs can promote complete fusion and hemifusion as alternative outcomes.

Using a cell fusion assay, we show here that in addition to complete fusion SNAREs also promote hemifusion as an alternative outcome. Approximately 65% of events resulted in full fusion, and the remaining 35% in hemifusion; of those, approximately two thirds were permanent and approximately one third were reversible. We predict that this relatively close balance among outcomes could be tipped by binding of regulatory proteins to the SNAREs, allowing for dynamic physiological regulation between full fusion and reversible kiss-and-run-like events.

3T3 Cells↗

PubMed2005

Concerted auto-regulation in yeast endosomal t-SNAREs.

In yeast, the assembly of the target (t)-SNAREs [Tlg2p/Tlg1p,Vti1p] and [Pep12p/Tlg1p,Vti1p] with the vesicular (v)-SNARE Snc2p promotes endocytic fusion. Here, selected mutations and truncations of SNARE proteins were tested in an in vitro fusion assay to identify potential regulatory regions in these proteins, and two distinct regions were found. The first is represented by the combined effect of the three t-SNARE N-terminal regions and the second is located within the Tlg1p SNARE motif. These internal controls provide a potential mechanism to enable SNARE-dependent fusion to be regulated.

Amino Acid Motifs↗

PubMed2005

Differential use of endoplasmic reticulum membrane for phagocytosis in J774 macrophages.

Sustained phagocytosis requires the continuous replacement of cell-surface membrane from intracellular sources. Depending on the nature of the engulfed particles, a variety of endocytic compartments have been demonstrated to contribute membranes needed for the formation of phagosomes. It has recently been reported that the endoplasmic reticulum (ER) can also fuse with the plasma membrane during phagocytosis [Gagnon, E., Duclos, S., Rondeau, C., Chevet, E., Cameron, P. H., Steele-Mortimer, O., Paiement, J., Bergeron, J. J. & Desjardins, M. (2002) Cell 110, 119-131]. However, there is currently no known mechanistic basis for this fusion process to occur. Here we report that direct ER-plasma membrane fusion during phagocytosis requires the ER resident soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein ERS24/Sec22b and that J774-macrophages react toward the challenge of large (3.0-microm) but not small (0.8-microm) particles by triggering this fusion mechanism, allowing them to access the most abundant endogenous membrane source in the cell, the ER.

Animals↗

PubMed2005

Sequence characteristics of functional siRNAs.

RNA interference in mammalian cells is actively used to conduct genetic screens, to identify and to validate targets, and to elucidate regulators and modifiers of cellular pathways. To ensure the specificity and efficacy of the active 21mer siRNA molecules, it is pertinent to develop a strategy for their rational design. Here we show that most functional siRNAs have characteristic sequence features. We tested 601 siRNAs targeting one exogenous and three endogenous genes. The efficacy of the siRNAs was determined at the protein level. Using a decision tree algorithm in combination with information analysis, our analyses revealed four sets of rules with a mean knockdown efficacy ranging from 60% to 73%. (To distinguish between percentages used to describe the quality of an siRNA and the percentages used to describe parts of data sets we underlined the former throughout this paper.) The best rule comprises an A/U at positions 10 and 19, a G/C at position 1, and more than three A/Us between positions 13 and 19, in the sense strand of the siRNA sequence. Using these rules, there is a 99.9% chance of designing an effective siRNA in a set of three with more than 50% knockdown efficiency in a biological readout.

Base Sequence↗

PubMed2004

Imaging single membrane fusion events mediated by SNARE proteins.

Using total internal reflection fluorescence microscopy, we have developed an assay to monitor individual fusion events between proteoliposomes containing vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and a supported planar bilayer containing cognate target SNAREs. Approach, docking, and fusion of individual vesicles to the target membrane were quantified by delivery and subsequent lateral spread of fluorescent phospholipids from the vesicle membrane into the target bilayer. Fusion probability was increased by raising divalent cations (Ca2+ and Mg2+). Fusion of individual vesicles initiated in <100 ms after the rise of Ca2+ and membrane mixing was complete in 300 ms. Removal of the N-terminal H(abc) domain of syntaxin 1A increased fusion probability >30-fold compared to the full-length protein, but even in the absence of the H(abc) domain, vesicle fusion was still enhanced in response to Ca2+ increase. Our observations establish that the SNARE core complex is sufficient to fuse two opposing membrane bilayers at a speed commensurate with most membrane fusion processes in cells. This real-time analysis of single vesicle fusion opens the door to mechanistic studies of how SNARE and accessory proteins regulate fusion processes in vivo.

Base Sequence↗

PubMed2004

Localization and activity of the SNARE Ykt6 determined by its regulatory domain and palmitoylation.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze compartment-specific membrane fusion. Whereas most SNAREs are bona fide type II membrane proteins, Ykt6 lacks a proteinaceous membrane anchor but contains a prenylation consensus motif (CAAX box) and exists in an inactive cytosolic and an active membrane-bound form. We demonstrate that both forms are farnesylated at the carboxyl-terminal cysteine of the CCAIM sequence. Farnesylation is the prerequisite for subsequent palmitoylation of the upstream cysteine, which permits stable membrane association of Ykt6. The double-lipid modification and membrane association is crucial for intra-Golgi transport in vitro and cell homeostasis/survival in vivo. The membrane recruitment and palmitoylation is controlled by the N-terminal domain of Ykt6, which interacts with the SNARE motif, keeping it in an inactive closed conformation. Together, these results suggest that conformational changes control the lipid modification and function of Ykt6. Considering the essential and central role of Ykt6 in the secretory pathway, this spatial and functional cycle might provide a mechanism to regulate the rate of intracellular membrane flow.

Cytosol↗

PubMed2004

The specificity of SNARE-dependent fusion is encoded in the SNARE motif.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the core of the fusion machinery, and isolated SNAREs fuse membranes with exquisite specificity by cognate pairing. Most SNAREs have a membrane-spanning region, an N-terminal domain, and a membrane proximal SNARE motif domain. Although the SNARE motif is critical for SNARE complex formation, is it the sole determinant of the specificity of SNARE-dependent fusion? To test this, we make use of a SNARE complex functioning in the late endosomal compartment in yeast. Studying this complex and the previously identified early endosomal SNARE complex, we find that the specificity of fusion resides in the SNARE motifs.

Amino Acid Motifs↗

PubMed2004

Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack.

Genetic and biochemical evidence has established that a SNARE complex consisting of syntaxin 5 (Sed5)-mYkt6 (Ykt6)-GOS28 (Gos1)-GS15 (Sft1) is required for transport of proteins across the Golgi stack in animals (yeast). We have utilized quantitative immunogold labeling to establish the cis-trans distribution of the v-SNARE GS15 and the t-SNARE subunits GOS28 and syntaxin 5. Whereas the distribution of the t-SNARE is nearly even across the Golgi stack from the cis to the trans side, the v-SNARE GS15 is present in a gradient of increasing concentration toward the trans face of the stack. This contrasts with a second distinct SNARE complex, also required for intra-Golgi transport, consisting of syntaxin 5 (Sed5)-membrin (Bos1)-ERS24 (Sec22)-rBet1 (Bet1), whose v-(rBet1) and t-SNARE subunits (membrin and ERS24), progressively decrease in concentration toward the trans face. Transport within the stack therefore appears to utilize countercurrent gradients of two Golgi SNAREpins and may involve a mechanism akin to homotypic fusion.

Animals↗

PubMed2003

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion.

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

Binding Sites↗

PubMed2003

A SNARE required for retrograde transport to the endoplasmic reticulum.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells. Sequence analysis of the yeast genome revealed a previously uncharacterized SNARE, SNARE-like tail-anchored protein 1 (Slt1). Slt1 is an essential protein localized in the endoplasmic reticulum (ER). It forms a SNARE complex with Sec22 and the ER syntaxin Ufe1. Down-regulation of Slt1 levels leads to improper secretion of proteins normally resident in the ER. We suggest that Slt1 is a component of the SNAREpin required for retrograde traffic to the ER. Based on the previously reported association with Ufe1 and Sec22, Sec20 likely contributes the fourth SNARE to the SNAREpin.

Amino Acid Sequence↗

PubMed2003

Fusion of cells by flipped SNAREs.

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) hypothesis suggests that pairs of proteins known as vesicle (v-) SNAREs and target membrane (t-) SNAREs interact specifically to control and mediate intracellular membrane fusion events. Here, cells expressing the interacting domains of v- and t-SNAREs on the cell surface were found to fuse spontaneously, demonstrating that SNAREs are sufficient to fuse biological membranes.

Animals↗