In pancreatic ␤-cells, the predominant voltage-gated Ca 2؉ channel (Ca V 1.2) and K ؉ channel (K V 2.1) are directly coupled to SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor) proteins. These SNARE proteins modulate channel expression and gating and closely associate these channels with the insulin secretory vesicles. We show that K V 2.1 and Ca V 1.2, but not K V 1.4, SUR1, or Kir6.2, target to specialized cholesterol-rich lipid raft domains on ␤-cell plasma membranes. Similarly, the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2, but not Munc-13-1 or n-Sec1, are associated with lipid rafts. Disruption of the lipid rafts by depleting membrane cholesterol with methyl-␤-cyclodextrin shunts K V 2.1, Ca V 1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-␤-cyclodextrin inhibits K V 2.1 but not Ca V 1.2 channel activity and enhances single-cell exocytic events and insulin secretion. Membrane compartmentalization of ion channels and SNARE proteins in lipid rafts may be critical for the temporal and spatial coordination of insulin release, forming what has been described as the excitosome complex.In the pancreatic islets of Langerhans, glucose uptake by ␤-cells initiates a cascade of cellular events resulting in insulin secretion. A key response leading to insulin release is the change in transmembrane potential associated with the opening and closing of ion channels. Glucose uptake and metabolism increases the ratio of ATP/ADP, leading to the blockade of ATP-sensitive potassium (K ϩ -ATP) channels. Inhibition of these channels results in cell membrane depolarization and subsequent activation of voltage-gated Ca 2ϩ (Ca V ) 1 channels. Influx of extracellular Ca 2ϩ through Ca V channels causes oscillatory elevations in [Ca 2ϩ ] i , fusion of insulin-containing vesicles with the cell membrane, and insulin release (reviewed in Ref. 1). This entire process is suppressed or terminated by the opening of voltage-gated K ϩ (K V ) channels (2). The integrated process of channel gating is critical for the coordination of insulin release and thus the consequent maintenance of proper plasma glucose levels.Pancreatic ␤-cells and clonal insulinoma cells express four different families of K V channels (K V 1, K V 2, K V 3, K V 4) in variable levels (2-4). K V 2.1 is the most abundant K V channel isoform expressed in both isolated islet ␤-cells and insulinoma cells. To support this notion, the dominant-negative knockout of K V 2.1 channel or pharmacological blockade with a selective K V 2.1 antagonist reduces steady-state outward K V currents by ϳ60 -70% (2, 5). In addition to K V 2.1, other K V channel ␣ subunits are expressed in pancreatic ␤-cells to a lesser extent, including K V 1.4 and K V 1.6, which account for less than 25% of outward K ϩ currents measured in these cells (2). The central role of Ca V channels in insulin secretion is well recognized (1). The predominant Ca V channel in ␤-cells is the L-type channel (long-lasting; Ca V 1.2/␣ 1C-a and Ca V 1.3/␣ 1D ) (6, 7). T...

Acquisition of additional genetic and/or epigenetic abnormalities other than the BCR/ABL fusion gene is believed to cause disease progression in chronic myeloid leukemia (CML) from chronic phase to blast crisis (BC). To gain insights into the underlying mechanisms of progression to BC, we screened DNA samples from CML patients during blast transformation for mutations in a number of transcription factor genes that are critical for myeloid-lymphoid development. In 85 cases of CML blast transformation, we identified two new mutations in the coding region of GATA-2, a negative regulator of hematopoietic stem/progenitor cell differentiation. A L359V substitution within zinc finger domain (ZF) 2 of GATA-2 was found in eight cases with myelomonoblastic features, whereas an in-frame deletion of 6 aa (⌬341-346) spanning the C-terminal border of ZF1 was detected in one patient at myeloid BC with eosinophilia. Further studies indicated that L359V not only increased transactivation activity of GATA-2 but also enhanced its inhibitory effects on the activity of PU.1, a major regulator of myelopoiesis. Consistent with the myelomonoblastic features of CML transformation with the GATA-2 L359V mutant, transduction of the GATA-2 L359V mutant into HL-60 cells or BCR/ABL-harboring murine cells disturbed myelomonocytic differentiation/proliferation in vitro and in vivo, respectively. These data strongly suggest that GATA-2 mutations may play a role in acute myeloid transformation in a subset of CML patients.blast crisis ͉ chronic phase ͉ genetic alteration ͉ transcriptional regulation

Voltage-gated K؉ (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet ␤-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet ␤-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet ␤-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634 -853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca 2؉ channels. In contrast to Ca 2؉ channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.

Purpose: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present inT-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients withT-ALL. Experimental Design: Detection of NOTCH1 mutation was done using reverse transcription-PCR amplification and direct sequencing, and thereby compared according to the clinical/ biological data of the patients. Results: Thirty-two mutations were identified in 29 patients (with dual mutations in 3 cases), involving not only the heterodimerization and proline/glutamic acid/serine/threonine domains as previously reported but also the transcription activation and ankyrin repeat domains revealed for the first time. These mutations were significantly associated with elevated WBC count at diagnosis and independently linked to short survival time. Interestingly, the statistically significant difference of survival according to NOTCH1 mutations was only observed in adult patients (>18 years) but not in pediatric patients (V18 years), possibly due to the relatively good overall response of childhoodT-ALL to the current chemotherapy. NOTCH1 mutations could coexist with HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation on prognosis was potentiated by HOX11L2 but was attenuated by HOX11. Conclusion: NOTCH1 mutation is an important prognostic marker in T-ALL and its predictive value could be even further increased if coevaluated with other T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with oncogenic transcriptional factors on T-ALL pathogenesis.

N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc 2 -PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc 2 -PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor.Asparagine (N)-glycosylation is an essential modification that regulates protein folding and stability. Prior to its attachment to protein, the oligosaccharide Glu 3 Man 9 GlcNAc 2 is assembled on the lipid carrier, dolichyl pyrophosphate (dol-PP), in the ER 2 (see Refs. 1-4 for review). The earliest steps of this lipid-linked oligosaccharide (LLO) synthesis begin on the cytoplasmic face of the ER. Seven sugars, (two N-acetylglucosamines and five mannoses) are sequentially added to dol-P to form Man 5 GlcNAc 2 -PP-dol by enzymes that have their catalytic domain on the cytosolic side of the ER membrane and use sugar nucleotide substrates (2, 5, 6). The enzymes that catalyze addition of the next seven sugars (four mannoses and three glucoses) do so within the lumen of the ER and use dolichol-linked sugar substrates (see Ref. 1 for review). Once assembled, this core oligosaccharide is transferred to protein by oligosaccharyltransferase through an N-glycosidic bond to an asparagine that is part of the Asn-X-(Ser/Thr) consensus sequence (7). Proteinlinked oligosaccharide is immediately modified by the removal of glucoses and mannose by ER glucosidases and mannosidases. Failure to properly synthesize, transfer, or modify the N-linked glycan results in glycoproteins that are recognized by the quality control systems that restrict these abe...

The slower kinetics of insulin release from pancreatic islet beta cells, as compared with other regulated secretory processes such as chromaffin granule secretion, can in part be explained by the small number of the insulin granules that are docked to the plasma membrane and readily releasable. In type-2 diabetes, the kinetics of insulin secretion become grossly distorted, and, to therapeutically correct this, it is imperative to elucidate the mechanisms that regulate priming and secretion of insulin secretory granules. Munc13-1, a synaptic protein that regulates SNARE complex assembly, is the major protein determining the priming of synaptic vesicles. Here, we demonstrate the presence of Munc13-1 in human, rat, and mouse pancreatic islet beta cells. Expression of Munc13-1, along with its cognate partners, syntaxin 1a and Munc18a, is reduced in the pancreatic islets of type-2 diabetes non-obese Goto-Kakizaki and obese Zucker fa/fa rats. In insulinoma cells, overexpressed Munc13-1-enhanced green fluorescent protein is translocated to the plasma membrane in a temperature-dependent manner. This, in turn, greatly amplifies insulin exocytosis as determined by patch clamp capacitance measurements and radioimmunoassay of the insulin released. The potentiation of exocytosis by Munc13-1 is dependent on endogenously produced diacylglycerol acting on the overexpressed Munc13-1 because it is blocked by a phospholipase C inhibitor (U73122) and abrogated when the diacylglycerol binding-deficient Munc13-1 H567K mutant is expressed instead of the wild type protein. Our data demonstrate that Munc13-mediated vesicle priming is not restricted to neurotransmitter release but is also functional in insulin secretion, where it is subject to regulation by the diacylglycerol second messenger pathway. In view of our findings, Munc13-1 is a potential drug target for therapeutic optimization of insulin secretion in diabetes.

The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1 gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in the gda1 null mutant caused a significant increase in microsomal membranebound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ ATP, GDP/GTP, and UDP/UTP and ϳ50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The ⌬ynd1 mutant cells were defective in O-and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in ⌬gda1 disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the ⌬ynd1⌬gda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells.

The substrates for glycan synthesis in the lumen of the Golgi are nucleotide sugars that must be transported from the cytosol by specific membrane-bound transporters. The principal nucleotide sugar used for glycosylation in the Golgi of the yeast Saccharomyces cerevisiae is GDP-mannose, whose lumenal transport is mediated by the VRG4 gene product. As the sole provider of lumenal mannose, the Vrg4 protein functions as a key regulator of glycosylation in the yeast Golgi. We have undertaken a functional analysis of Vrg4p as a model for understanding nucleotide sugar transport in the Golgi. Here, we analyzed epitope-tagged alleles of VRG4. Gel filtration chromatography and co-immunoprecipitation experiments demonstrate that the Vrg4 protein forms homodimers with specificity and high affinity. Deletion analyses identified two regions essential for Vrg4p function. Mutant Vrg4 proteins lacking the predicted C-terminal membrane-spanning domain fail to assemble into oligomers (Abe, M., Hashimoto, H., and Yoda, K. (1999) FEBS Lett. 458, 309 -312) and are unstable, while proteins lacking the N-terminal cytosolic tail are stable and multimerize efficiently, but are mislocalized to the endoplasmic reticulum (ER). Fusion of the N terminus of Vrg4p to related ER membrane proteins promote their transport to the Golgi, suggesting that sequences in the N terminus supply information for ER export. The dominant negative phenotype resulting from overexpression of truncated Vrg4-⌬N proteins provides strong genetic evidence for homodimer formation in vivo. These studies are consistent with a model in which Vrg4p oligomerizes in the ER and is subsequently transported to the Golgi via a mechanism that involves positive sorting rather than passive default.The Golgi complex serves as the intracellular site for the terminal carbohydrate modifications of proteins and lipids. These modifications are essential for life and play a variety of important biological roles, from protein folding to the regulation of cell surface properties. The substrates for the carbohydrate modification of both glycoproteins and glycolipids in the Golgi are nucleotide sugars, whose site of synthesis is the cytosol. These molecules must be transported into the Golgi lumen by membrane-bound nucleotide sugar transporters (NSTs) 1 to be utilized by the glycosyltransferases. The current model for the transport of nucleotide sugars by the NSTs involves a one-for-one exchange reaction, in which the lumenal transport of a nucleotide sugar from the cytoplasm is coupled to the equimolar exit of the corresponding nucleotide monophosphate (2-4). The nucleotide monophosphate is generated through the action of the glycosyltransferases and nucleoside diphosphatases in the lumen of the Golgi. As a consequence of their role in substrate provision, the NSTs play an indispensable role in glycoconjugate synthesis, best evidenced by the severe phenotype of mutants with defects in Golgi transport of nucleotide sugars (for review, see Refs. 5 and 6).Many NST activities have been reporte...