Proteolytic Processing of the γ-Subunit Is Associated with the Failure to Form GlcNAc-1-phosphotransferase Complexes and Mannose 6-Phosphate Residues on Lysosomal Enzymes in Human Macrophages

GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, α₂β₂γ₂, that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3–10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the γ-subunit but not the β-subunit was found to be proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the γ-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that proteolytic processing of the γ-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.     

Compensatory expression of human N-Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma

The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markers essential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (α₂, β₂, γ₂). The α- and β-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the γ-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GNPTG cause mucolipidosis type III gamma, which is characterized by missorting and cellular loss of lysosomal enzymes leading to lysosomal accumulation of storage material. Using plasmon resonance spectrometry, we showed that recombinant γ-subunit failed to bind the lysosomal enzyme arylsulfatase A. Additionally, the overexpression of the γ-subunit in COS7 cells did not result in hypersecretion of newly synthesized lysosomal enzymes expected for competition for binding sites of the endogenous phosphotransferase complex. Analysis of fibroblasts exhibiting a novel mutation in GNPTG (c.619insT, p.K207IfsX7) revealed that the expression of GNPTAB was increased whereas in γ-subunit overexpressing cells the GNPTAB mRNA was reduced. The data suggest that the γ-subunit is important for the balance of phosphotransferase subunits rather for general binding of lysosomal enzymes.     

Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport

GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2β2γ2 hexamers in which the core catalytic α- and β-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP–glucose glycoprotein glucosyltransferase. Comparison with UDP–glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.     

Transport of the GlcNAc-1-phosphotransferase α/β-Subunit Precursor Protein to the Golgi Apparatus Requires a Combinatorial Sorting Motif

The Golgi-resident N-acetylglucosamine-1-phosphotransferase (PT) complex is composed of two α-, β-, and γ-subunits and represents the key enzyme for the biosynthesis of mannose 6-phosphate recognition marker on soluble lysosomal proteins. Mutations in the PT complex cause the lysosomal storage diseases mucolipidosis II and III. A prerequisite for the enzymatic activity is the site-1 protease-mediated cleavage of the PT α/β-subunit precursor protein in the Golgi apparatus. Here, we have investigated structural requirements of the PT α/β-subunit precursor protein for its efficient export from the endoplasmic reticulum (ER). Both wild-type and a cleavage-resistant type III membrane PT α/β-subunit precursor protein are exported whereas coexpressed separate α- and β-subunits failed to reach the cis-Golgi compartment. Mutational analyses revealed combinatorial, non-exchangeable dileucine and dibasic motifs located in a defined sequence context in the cytosolic N- and C-terminal domains that are required for efficient ER exit and subsequent proteolytic activation of the α/β-subunit precursor protein in the Golgi. In the presence of a dominant negative Sar1 mutant the ER exit of the PT α/β-subunit precursor protein is inhibited indicating its transport in coat protein complex II-coated vesicles. Expression studies of missense mutations identified in mucolipidosis III patients that alter amino acids in the N- and C-terminal domains demonstrated that the substitution of a lysine residue in close proximity to the dileucine sorting motif impaired ER-Golgi transport and subsequent activation of the PT α/β-subunit precursor protein. The data suggest that the oligomeric type III membrane protein PT complex requires a combinatorial sorting motif that forms a tertiary epitope to be recognized by distinct sites within the coat protein complex II machinery.     

The Pharmacology of the Benzodiazepine Site of the GABA-A Receptor is Dependent on the Type of γ-Subunit Present

Abstract

The pharmacology of native and recombinant GABA-A receptors containing either γ1, γ2 or γ3 subunits has been investigated. The pharmacology of native receptors has been investigated by immunoprecipitating receptors from solubilised preparations of rat brain with antisera specific for individual γ-subunits and analysing their radioligand binding characteristics. Receptors containing a γ1-subunit do not bind benzodiazepine radioligands with high affinity. Those containing either a γ2 or γ3 subunit bind [³H]flumazenil with high affinity. Some compounds compete for these binding sites with multiple affinities, reflecting the presence of populations of receptors containing several different types of α-subunit. Photoaffinity-labelling of GABA-A receptors from a cell line stably expressing GABA-A receptors of composition α₁β3γ2 followed by immunoprecipitation of individual subunits revealed that the α and γ but not the β-subunit could be irreversibly labelled by [³H]flunitrazepam.

The properties of recombinant receptors have been investigated in oocytes expressing γ1, γ2, or γ3 subunits in combination with an α and a β-subunit. Some compounds such as zolpidem, DMCM and flunitrazepam show selectivity for receptors containing different γ-subunits. Others such as CL 218,872 show no selectivity between receptors containing different γ-subunits but exhibit selectivity for receptors containing different α-subunits. These data taken together suggest that the benzodiazepine site of the GABA-A receptor is formed with contributions from both the α and γ-subunits.     

Site-1 protease-activated formation of lysosomal targeting motifs is independent of the lipogenic transcription control

Site-1 protease (S1P) cleaves membrane-bound lipogenic sterol regulatory element-binding proteins (SREBPs) and the α/β-subunit precursor protein of the N-acetylglucosamine-1-phosphotransferase forming mannose 6-phosphate (M6P) targeting markers on lysosomal enzymes. The translocation of SREBPs from the endoplasmic reticulum (ER) to the Golgi-resident S1P depends on the intracellular sterol content, but it is unknown whether the ER exit of the α/β-subunit precursor is regulated. Here, we investigated the effect of cholesterol depletion (atorvastatin treatment) and elevation (LDL overload) on ER-Golgi transport, S1P-mediated cleavage of the α/β-subunit precursor, and the subsequent targeting of lysosomal enzymes along the biosynthetic and endocytic pathway to lysosomes. The data showed that the proteolytic cleavage of the α/β-subunit precursor into mature and enzymatically active subunits does not depend on the cholesterol content. In either treatment, lysosomal enzymes are normally decorated with M6P residues, allowing the proper sorting to lysosomes. In addition, we found that, in fibroblasts of mucolipidosis type II mice and Niemann-Pick type C patients characterized by aberrant cholesterol accumulation, the proteolytic cleavage of the α/β-subunit precursor was not impaired. We conclude that S1P substrate-dependent regulatory mechanisms for lipid synthesis and biogenesis of lysosomes are different.     

Thermodynamic analysis reveals that GTP binding affects the interaction between the alpha- and gamma-subunits of translation initiation factor 2

Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of α-, β-, and γ-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the α- and γ-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The α-subunits bound to the γ-subunit with large heat capacity change (ΔC_p) values. The ΔH and ΔC_p values for the interaction between the α- and γ-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the α-subunit and GTP changes the conformation of the switch region of the γ-subunit and increases the affinity of the γ-subunit for tRNA.     

GNPTAB missense mutations cause loss of GlcNAc-1-phosphotransferase activity in mucolipidosis type II through distinct mechanisms

Mucolipidoses (ML) II and III alpha/beta are lysosomal storage diseases caused by pathogenic mutations in GNPTAB encoding the α⁄β-subunit precursor of GlcNAc-1-phosphotransferase. To determine genotype-phenotype correlation and functional analysis of mutant GlcNAc-1-phosphotransferase, 13 Brazilian patients clinically and biochemical diagnosed for MLII or III alpha/beta were studied. By sequencing of genomic GNPTAB of the MLII and MLIII alpha/beta patients we identified six novel mutations: p.D76G, p.S385L, p.Q278Kfs*3, p.H588Qfs*27, p.N642Lfs*10 and p.Y1111*. Expression analysis by western blotting and immunofluorescence microscopy revealed that the mutant α⁄β-subunit precursor p.D76G is retained in the endoplasmic reticulum whereas the mutant p.S385L is correctly transported to the cis-Golgi apparatus and proteolytically processed. Both mutations lead to complete loss of GlcNAc-1-phosphotransferase activity, consistent with the severe clinical MLII phenotype of the patients. Our study expands the genotypic spectrum of MLII and provides novel insights into structural requirements to ensure GlcNAc-1-phosphotransferase activity.     

Structural homology among the major 7s globulin subunits of soybean seed storage proteins

The soybean seed 7S globulin subunits, i.e. α, α′, β and γ-subunits of β-conglycinin, the γ-conglycinin subunit and the HI/HII and LII subunits of basic 7S globulin were purified and the NH₂-terminal amino acid sequences of all these subunits except the γ-subunit of β-conglycinin were determined. Only the NH₂-terminal regions of the α and α′-subunits showed high sequence homology. However, sequencing of tryptic peptides from the seven subunits revealed that internal region sequences were highly homologous among the four subunits of β-conglycinin. In contrast to the β-conglycinin subunits, no sequence homology was found among the other subunits. On the basis of these results, the major 7S globulin fraction is considered more heterogeneous in primary structure than another major globulin fraction, 11S globulin (glycinin), in soybean seeds.     

Identification of heterotrimeric G proteins in human sperm tail membranes

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein α-subunits, and A 86, which detects all known pertussis toxin-sensitive α-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein α-subunits failed to detect any specific antigens in enriched tail membranes AS 36, recognizing the ã2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa β₁-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α-subunits nor G protein β-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive α-subunits. However, membrane preparations of nonpurified human spermatozoa contained α₂ subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein α-subunits; the putative β-subunit was identified as a β₂-subunit. © 1995 Wiley-Liss, Inc.     

Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries

1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, α-subunit 40600, β-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the α-subunit of the decarboxylase. 5. The molar ratio of phosphate to α-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [³²P]phosphate complex by using methyl [1-¹⁴C]acetimidate, followed by separation of α-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two α-chains (i.e. one phosphate group/α₂β₂ tetramer). Complete phosphorylation corresponded to three phosphate groups per α₂β₂ tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 α-subunits:4 β-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.     

The isolation and amino acid sequence of the β- and γ-subunits of the lectin from the seeds of Dioclea Grandiflora

Although the two smaller β- and γ- subunits of the lectin from Dioclea grandiflora were clearly resolved by sodium dodecyl sulphate (SDS) gel electrophoresis, the concensus of other techniques including ultracentrifugation, isoelectric focusing in 8 M urea, size-exclusion chromatography in dissociating solvents and amino acid and sequence analysis indicated that they were similar in molecular size and that they had arisen either by a single enzymic cleavage at Asn¹¹⁸-Ser¹¹⁹ in the middle of the 237 residue-long mature α-subunit or by multiple cleavages occurring during post-translational processing of intermediates. The existence of minor forms of the β- and γ- subunits resulting from a cleavage at Asn¹²⁴-Ser¹²⁵ of the α-subunit was also recognized. The results indicated that the apparent difference in molecular size of the β- and γ-subunits deduced from SDS-gel electrophoresis could be explained by the anomalous behaviour of both subunits in this separation technique. The structural features of the D. grandiflora lectin are compared with those of concanavalin A obtained from seeds of the botanically related Canavalia ensiformis.     

Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase

Mucolipidosis II (ML II) is a fatal lysosomal storage disorder resulting from defects in the multimeric GlcNAc-1-phosphotransferase responsible for the initial step in the generation of the mannose 6-phosphate (M6P) recognition marker. M6P residues on oligosaccharides of newly synthesized lysosomal enzymes are essential for efficient receptor-mediated transport to lysosomes. We used the recombinant GlcNAc-1-phosphotransferase gamma subunit as an affinity matrix to purify an unknown protein identified as the product of GNPTA (encoding GNPTA, previously known as MGC4170). The cDNA encodes a protein of 1,256 amino acids with two putative transmembrane domains and a complex preserved modular structure comprising at least six domains. The N-terminal domain of GNPTA, interrupted by a long insertion, shows similarities to bacterial capsule biosynthesis proteins. We identified seven mutations in GNPTA that lead to premature translational termination in six individuals with ML II. Retroviral transduction of fibroblasts from an individual with ML II resulted in the expression and localization of GNPTA in the Golgi apparatus, accompanied by the correction of hypersecretion of lysosomal enzymes. Our results provide evidence that GNPTA encodes a subunit of GlcNAc-1-phosphotransferase defective in individuals with ML II.     

Crystal structure of the intact archaeal translation initiation factor 2 demonstrates very high conformational flexibility in the alpha- and beta-subunits

In Eukarya and Archaea, translation initiation factor 2 (eIF2/aIF2), which contains three subunits (α, β, and γ), is pivotal for binding of charged initiator tRNA to the small ribosomal subunit. The crystal structure of the full-sized heterotrimeric aIF2 from Sulfolobus solfataricus in the nucleotide-free form has been determined at 2.8-Å resolution. Superposition of four molecules in the asymmetric unit of the crystal and the comparison of the obtained structures with the known structures of the aIF2αγ and aIF2βγ heterodimers revealed high conformational flexibility in the α- and β-subunits. In fact, the full-sized aIF2 consists of a rigid central part, formed by the γ-subunit, domain 3 of the α-subunit, and the N-terminal α-helix of the β-subunit, and two mobile “wings,” formed by domains 1 and 2 of the α-subunit, the central part, and the zinc-binding domain of the β-subunit. High structural flexibility of the wings is probably required for interaction of aIF2 with the small ribosomal subunit. Comparative analysis of all known structures of the γ-subunit alone and within the heterodimers and heterotrimers in nucleotide-bound and nucleotide-free states shows that the conformations of switch 1 and switch 2 do not correlate with the assembly or nucleotide states of the protein.     

Molecular characterization and evolution of haemocyanin from the two freshwater shrimps Caridina multidentata (Stimpson, 1860) and Atyopsis moluccensis (De Haan, 1849)

Haemocyanin (Hc) is a copper-containing respiratory protein, floating freely dissolved in the hemolymph of many arthropod species. A typical haemocyanin is a hexamer or oligohexamer of six identical or similar subunits, with a molecular mass around 75 kDa each. In the crustaceans, the haemocyanins appear to be restricted to the remipedes and the malacostracans. We have investigated the haemocyanins of two freshwater shrimps, the Amano shrimp Caridina multidentata and the bamboo shrimp Atyopsis moluccensis. We obtained three full-length and one partial cDNA sequences of haemocyanin subunits from the Amano shrimp, which were assigned to the α- and γ-types of decapod haemocyanin subunits. Three complete and two partial haemocyanin cDNA sequences were obtained from the bamboo shrimp, which represent subunit types α, β and γ. This is the first time that sequences of all three subunit types of the decapod haemocyanins were obtained from a single species. However, mass spectrometry analyses identified only α- and γ-type subunits, suggesting that a β-subunit is not a major component of the native haemocyanin of the bamboo shrimp. Phylogenetic and molecular clock analyses showed that malacostracan haemocyanins commenced to diversify into distinct subunit types already ~515 million years ago. β-subunits diverged first, followed by α- and γ-type subunits ~396 million years ago. The haemocyanins of phyllocarids and peracarids form distinct clades within the α/γ-cluster. Within the Caridea, an early divergence of distinct α-type subunits occurred ~200 MYA. The tree of the γ-subunits suggests a common clade of the Caridea (shrimps) and Penaeidae (prawns).     

Molecular Architecture of Photoreceptor Phosphodiesterase Elucidated by Chemical Cross-Linking and Integrative Modeling

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αβγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αβ), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for differential binding of the γ-subunit to the GAFa domains of the α- and β-subunits are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.     

Rotary catalysis of the stator ring of F(1)-ATPase

F(1)-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α(3)β(3) ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a "dictator" completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F(1)-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Polarized membrane distribution of potassium-dependent ion pumps in epithelial cells: Different roles of the <Emphasis Type="Italic">N</Emphasis>-glycans of their <Emphasis Type="Italic">β</Emphasis> subunits

The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P₂-type pumps that catalyze active transport of Na⁺ in exchange for K⁺ (Na,K-ATPase) or H⁺ in exchange for K⁺ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic α-subunit and a glycosylated β-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the β-subunits of the Na,K-ATPase and H,K-ATPase are important for stable membrane integration of their respective α subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled β-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the␣maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the β-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase β ₁-subunit is the major β-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase β ₁-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the β ₁-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the α-subunit to the cytoskeleton. This intercellular β ₁–β ₁ interaction is also important for formation of cell–cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase β ₂-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the β ₂-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase β-subunit determine apical sorting of this subunit. Supported in part by NIH grants DK46917, DK58333, D53642, and USVA