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PubMed2003

Imprint of somatic hypermutation differs in human immunoglobulin heavy and lambda chain variable gene segments.

Somatic hypermutation (SHM) introduces mutations into immunoglobulin (Ig) variable gene segments, thus diversifying the B cell repertoire prior to positive selection of high affinity variants during maturation of T cell-dependent B cell responses. Somatic hypermutation of Ig heavy chain generates predominantly single base substitutions, favoring transition rather than transversion substitutions, and tends to direct mutations to specific 4-mer target sequences with G in second and C in third position. Here we have analyzed heavily mutated, nonproductively rearranged Ig lambda chain variable gene segments from human intestinal plasma cells, controlling for germline composition of the genes and local sequence variability. We have observed significant G.C strand bias in IgV(lambda), and differences in some di- and trinuleotide target preferences in IgV(lambda) compared to IgV(H). There is also a significant tendency to accumulate adjacent triplet mutations in IgV(lambda), which is not evident in IgV(H) in normal circumstances. These observations suggest that some aspect of the mechanism of somatic hypermutation operates differently in human immunoglobulin heavy and lambda light chain variable gene segments.

Aged↗

PubMed1998

Direct identification of a second distinct site of contact between cholecystokinin and its receptor.

We have developed a biologically active analogue of cholecystokinin (CCK) that incorporates a photolabile benzoylphenylalanine (Bpa) moiety in the middle of its pharmacophoric domain, which efficiently establishes a covalent bond with an interacting domain of the CCK receptor. This probe incorporated L-Bpa in the position of Gly29 of the well characterized, radioiodinatable CCK analogue, D-Tyr-Gly-[(Nle28,31)CCK-26-33]. It was a potent pancreatic secretagogue (EC50 = 28 +/- 6 nM) that was equally efficacious with natural CCK, and bound to the CCK receptor with moderate affinity (IC50 = 450 +/- 126 nM). This was adequate to allow specific covalent labeling of the receptor. The labeled domain was within the cyanogen bromide fragment of the receptor including the top of TM6 (the sixth transmembrane domain), the third extracellular loop, and TM7 (the seventh transmembrane domain), as proven by direct Edman degradation sequencing. When this fragment was modified by the replacement of Val342 with Met to generate an additional site of cyanogen bromide cleavage, the labeled fragment was reduced in apparent size consistent with its representing the carboxyl-terminal portion of this fragment. Radiochemical sequencing of that fragment demonstrated covalent attachment of the probe to His347 and Leu348 in this domain. This represents the second experimentally demonstrated contact between a CCK analogue and this receptor, complementing the labeling of the domain just above TM1 (the first transmembrane domain) by a photolabile residue at the carboxyl terminus of CCK (Ji, Z. S., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (1997) J. Biol. Chem. 272, 24393-24401). Both contacts are consistent with the conformational model of CCK binding proposed on the basis of the initial contact.

Amino Acid Sequence↗

PubMed1989

Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.

Amino Acid Sequence↗

PubMed2025

Sexual selection, genomic evolution and population fitness in Drosophila pseudoobscura.

Sexual selection shapes the genome in unique ways. It is also likely to have significant fitness consequences, such as purging deleterious mutations from the genome or conversely maintaining genetic load in a population via sexual conflict. Here, we examined what the influence of sexual selection has on genomic variation potentially underlying population fitness using experimentally evolved Drosophila pseudoobscura populations. Sexual selection was manipulated by keeping replicate lines in elevated polyandry or strict monogamy for approximately 200 generations followed by individual-based sequencing. Using pi (π), fixation index (Fst)and recombination rate measures, we confirmed signatures of selection were not dispersed but mainly localized to the third and X chromosome. Overall mutational load was similar between lines but our analysis of the distribution of fitness effects revealed considerable variation between lines and chromosomes. Furthermore, we found that the distribution of transposable elements differs between the lines, with a higher load in monogamous lines. Our results suggest that complex interactions between purifying selection and sexual conflict are shaping the genome, particularly on chromosome 3 and the sex chromosome; sexual selection influences divergence across chromosomes but in a more complex way than proposed by simple 'purging' of deleterious loci.

Animals↗

PubMed1989

Transforming activity of a 16-amino-acid segment of the bovine papillomavirus E5 protein linked to random sequences of hydrophobic amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the smallest transforming protein yet described. Previous results from our laboratory indicate that a hydrophobic core and specific carboxyl-terminal amino acids are required for the E5 protein to exert its transforming function. In this study, additional substitution mutations were generated in the E5 gene to determine the minimal amino acid sequence requirements for focus formation in mouse C127 cells. In most cases examined, substitution of the hydrophobic middle third of the E5 protein with unrelated hydrophobic sequences severely inhibited transforming activity. However, we have identified one hydrophobic amino acid sequence apparently unrelated to the wild-type one that can replace the middle third of the wild-type E5 protein without affecting the ability of the protein to stably transform cells or interact with cell membranes. Furthermore, a mutant E5 protein in which only the carboxyl-terminal 16 amino acids of the protein have been derived from E5 sequences retains transforming activity. Since several residues in the carboxyl-terminal portion of the E5 protein can be freely substituted with different amino acids (B. H. Horwitz, A. L. Burkhardt, R. Schlegel, and D. DiMaio, Mol. Cell. Biol. 8:4071-4078, 1988), the results reported here imply that much of the specific information necessary for cell transformation can be supplied by a subset of the carboxyl-terminal 16 amino acids of this protein.

Amino Acid Sequence↗

PubMed1986

Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions.

The plasma proteins, alpha 2-macroglobulin and complement components 3 and 4, contain an internal thiol ester involving a glutamyl and cysteinyl residue. The thiol ester is susceptible to cyclization at greater than 37 degrees C and forms an unstable 5-oxyproline intermediate. The latter can be hydrolyzed to produce two peptide fragments. We propose that enzymes having activated glutamyl residues as part of their catalytic mechanisms may undergo an analogous cyclization and peptidyl cleavage. As a model, we have investigated pig heart succinyl-CoA:3-keto acid transferase. When the CoA-enzyme thiolester intermediate is heated at pH 7.4 and 70 degrees C for 1 h, approximately 60% of the Mr = 60,000 subunits are cleaved to give Mr = 40,000 and 20,000 fragments. We have shown that formation of the enzyme thiolester is an obligate precursor for the protein fragmentation. However, the reaction was incomplete with a maximum of approximately 65% cleavage at times greater than 60 min. These results suggest that there is a competing, deactivation reaction; namely, the thiol ester and oxyproline intermediates are hydrolyzed to regenerate the active site glutamic acid. Although the maximum rate of cleavage is at 70 degrees C, approximately 15% autolysis also occurs at 37 degrees C. The Mr = 40,000 fragment had the same amino terminal sequence as the Mr = 60,000 subunit, (Trp-Lys-Phe-Tyr-Thr-Asp-Ala-Val-Glu-Ala-). No amino terminal could be detected for the Mr = 20,000 fragment, even after digesting the fragment with pyroglutaminase. Peptide maps of the fragments and the uncleaved subunit indicate that the fragments are generated in parallel. The size of the fragments puts the active site about two-thirds of the way from the amino terminal of the protein.

Acyltransferases↗

PubMed2000

Clinical, biochemical, and genetic characterization of a novel estrogen-dependent inherited form of angioedema.

BACKGROUND: Two genetic forms of hereditary angioedema (HAE) are currently recognized. Both are transmitted in an autosomal dominant manner and are characterized by recurrent episodes of localized angioedema. Involvement of the gut leads to episodes of severe abdominal pain, and laryngeal involvement can lead to airway obstruction and even death. One type results from heterozygosity for a nonexpressed C1 inhibitor allele, and the other results from heterozygosity for a nonfunctional C1 inhibitor allele. OBJECTIVE: This report identifies a third type of HAE, with a unique estrogen-dependent phenotype. METHODS: Detailed medical histories were obtained from family members, and a pedigree was constructed to ascertain the mode of inheritance. Determination of serum complement factors, C1 inhibitor protein, C1 inhibitor function, coagulation factor XII, plasma prekallikrein, high molecular weight kininogen, and selected DNA sequences were performed in affected members by using standard assays. RESULTS: Episodes of angioedema were clinically indistinguishable from those associated with previously described forms of HAE; however, these occurred only during pregnancy or the use of exogenous estrogens. Patients were otherwise asymptomatic, except for one patient who had acetyl salicylic acid/nonsteroidal anti-inflammatory drug-related angioedema later in life. History was available for members spanning 4 generations, and affected individuals were identified in 3 generations. Of 46 family members, phenotype could be determined in 13 members. Seven were affected, and 6 were not. One male of undetermined phenotype was an obligate carrier. The unique estrogen-dependent nature of the phenotype means that the status of several members in the third and fourth generation remains unknown. The disorder appears to be transmitted in an autosomal dominant fashion, although other modes of inheritance cannot be excluded entirely. C1 inhibitor protein, C1 inhibitor function, C2, C4, C1q, coagulation factor XII, prekallikrein, and high molecular kininogen were normal in 3 affected family members during asymptomatic periods. DNA sequencing revealed no abnormality in 3 patients in the coding region of the gene encoding C1 inhibitor or in the 5' flanking regions of the genes encoding C1 inhibitor and factor XII. CONCLUSIONS: This family appears to have a novel form of inherited angioedema that does not result from C1 inhibitor deficiency or dysfunction. The phenotype is uniquely estrogen dependent. Implications for diagnosis and treatment are discussed. Further studies are required to define the exact nature of the genetic abnormality involved.

Adult↗

PubMed1992

HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides.

The mutant human cell line T2 is defective in antigen presentation in the context of class I major histocompatibility complex (MHC) molecules, and also in that transfected T2 cells show poor surface expression of exogenous human class I (HLA) alleles. Both defects are thought to lie in the transport of antigenic peptides derived from cytosolic proteins into the endoplasmic reticulum (ER), as peptide-deficient class I molecules might be expected to be either unstable or retained in the ER. The products of several mouse class I (H-2) genes, and the endogenous gene HLA-A2 do, however, reach the surface of T2 cells at reasonable levels although they are non-functional. We report here that, as expected, poorly surface-expressed HLA molecules do not significantly bind endogenous peptides. Surprisingly, H-2 molecules expressed in T2 also lack associated peptides, arguing that 'empty' complexes of mouse class I glycoproteins with human beta 2-microglobulin are neither retained in the ER nor unstable. HLA-A2 molecules, however, do bind high levels of a limited set of endogenous peptides. We have sequenced three of these peptides and find that two, a 9-mer and an 11-mer, are derived from a putative signal sequence (of IP-30, an interferon-gamma-inducible protein), whereas a third, a 13-mer, is of unknown origin. The unusual length of two of the peptides argues that the 9-mers normally associated with HLA-A2 molecules may be generated before their transport from the cytosol rather than in a pre-Golgi compartment. To our knowledge, this is the first report of the isolation of a fragment of a eukaryotic signal peptide generated in vivo.

Alleles↗

PubMed2001

Safety considerations in vector development.

The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols. Because of limitations in animal models to evaluate lentiviral vectors for their potential to recombine and induce disease, the vector design itself should ensure against the emergence of RCR in vivo. Issues related to RCR generation and one approach to dealing with this problem are discussed in this chapter. To assess the risk of generating RCR, a highly sensitive biological assay was developed to specifically detect vector recombination in transduced cells. Analysis of lentiviral vector stocks has shown that recombination occurs during reverse transcription in primary target cells. Rejoining of viral protein-coding sequences of the packaging construct and cis-acting sequences of the vector was demonstrated to generate env-minus recombinants (LTR-gag-pol-LTR). Mobilization of recombinant lentiviral genomes was also demonstrated but was dependent on pseudotyping of the vector core with an exogenous envelope protein. 5' sequence analysis has demonstrated that recombinants consist of U3, R, U5, and the psi packaging signal joined with an open gag coding region. Analysis of the 3' end has mapped the point of vector recombination to the poly(A) tract of the packaging construct's mRNA. The state-of-the-art third generation packaging construct and SIN vector also have been shown to generate env-minus proviral recombinants capable of mobilizing retroviral DNA when pseudotyped with an exogenous envelope protein. A new class of HIV-based vector (trans-vector) was recently developed that splits the gag-pol component of the packaging construct into two parts: one that expresses Gag/Gag-Pro and another that expresses Pol (RT and IN) fused with Vpr. Unlike other lentiviral vectors, the trans-vector has not been shown to form recombinants capable of DNA mobilization. These results indicate the trans-vector design prevents the generation of env-minus recombinant lentivirus containing a functional gag-pol structure (LTR-gag-pol-LTR), which is absolutely required for retroviral DNA mobilization and the emergence of RCR. Quality assurance based on monitoring for RCR may have limitations as a predictor of safety in vivo, especially in the long term. The demonstration of lentivirus infection via alternative entry mechanisms supports this notion. Therefore, the approach of monitoring trans-vector stocks for env-minus recombinant virus in vitro as a surrogate marker for the possible emergence of RCR in vivo should represent a significant advancement in vector safety quality assurance.

Animals↗

PubMed1987

The nucleotide sequence of the gene encoding the F protein of canine distemper virus: a comparison of the deduced amino acid sequence with other paramyxoviruses.

The nucleotide sequence of the gene encoding the fusion protein of canine distemper virus was determined from cDNA clones derived from virus genome RNA and poly(A)+ RNA extracted from infected cells. The mRNA encoding the F protein is about 2300 nucleotides in length including the 3' poly(A) tail. There is a large open reading frame from nucleotides 86 to 2071 which begins at the first AUG codon in the F mRNA. This reading frame encodes a protein of 662 amino acid residues with a calculated mol. wt. of 73001. The first major hydrophobic domain in the amino acid sequence of the deduced protein (residues 104 to 130) may represent all or part of a signal sequence for cleavage of the N terminal part of the F2 protein. There are four potential N glycosylation sites in the F protein located within the F2 part of the molecule or the putative signal sequence, and one in the F1 portion. A second hydrophobic region corresponds to the proteolytic cleavage site which generates the F2 and F1 subunits. This stretches from residue 225 to 262 and the N terminal part of the F1 protein shows sequence conservation with the other paramyxoviruses. A third major hydrophobic domain near the C terminus of the F protein probably represents the membrane anchor for the F protein (residues 602 to 630). The F1 proteins of six paramyxoviruses are compared and shown to have substantial conservation of those residues important in the maintenance of tertiary structure of this protein.

Amino Acid Sequence↗

PubMed1989

Pelizaeus-Merzbacher disease: tight linkage to proteolipid protein gene exon variant.

Pelizaeus-Merzbacher disease (PMD) is a human X chromosome-linked dysmyelination disorder of the central nervous system for which the genetic defect has not yet been established. The jimpy mutation jp of the mouse is an X chromosome-linked disorder of myelin formation. The mutation is at an intron/exon splice site in the mouse gene for proteolipid protein (PLP). With the jimpy mouse mutation as a precedent, we focused our attention on the human PLP gene, which is found at Xq22. The polymerase chain reaction was used to amplify the exons of the PLP gene of an affected male from a large Indiana PMD kindred. DNA sequencing showed a C----T transition at nucleotide 40 of the second exon. An affected third cousin also showed this sequence variation, while two unaffected male relatives (sons of an obligate carrier female) had the normal cytidine nucleotide. Allele-specific oligonucleotides were used to generate data for linkage studies on the above mentioned PMD kindred. Our results show tight linkage (theta = 0) of PMD to PLP with a lod (logarithm of odds) score of 4.62. In six other unrelated PMD kindreds, only the normal-sequence oligonucleotide hybridized, which indicates genetic heterogeneity. The radical nature of the predicted amino acid change (proline to leucine), suggests that the PMD-causing defect may have been delineated in one kindred.

Base Sequence↗

PubMed1991

Escherichia coli molecular genetic map (1500 kbp): update II.

The DNA sequence data for Escherichia coli deposited in the EMBL library (release 27), together with miscellaneous data obtained from several laboratories, have been localized on an updated and corrected version of the restriction map of the chromosome generated by Kohara et al. (1987) and modified by others. This second update adds a further 500 kbp, increasing the amount of the E. coli chromosome sequenced to about one third of the total: 1510 kbp of sequenced DNA is included in the present data base. The accuracy of the map is assessed, and allows us to propose a precise genetic map position for every sequenced gene. The location of rare-cutting sites such as AvrII, NotI and SfiI have also been included in the update in order to combine the data obtained from different sources into one single file. The distribution of palindromic sequences (to which most restriction sites belong) has been studied in coding sequences. There appears to be a significant counter-selection against several such sequences in E. coli coding sequences (but not in other organisms such as Saccharomyces cerevisiae), suggesting the existence of constraints on DNA structure in E. coli, perhaps indicative of a functional role for horizontal gene transfer, preserving coding sequences, in this type of bacteria.

Base Sequence↗

PubMed2002

Novel homozygous splice acceptor site GnRH receptor (GnRHR) mutation: human GnRHR "knockout".

Mutations in the GnRH receptor (GnRHR) have been shown to be responsible for a significant number of autosomic recessive and, less commonly, sporadic cases of idiopathic hypogonadotropic hypogonadism. We describe a woman with complete GnRH resistance secondary to a novel homozygous GnRHR gene mutation, transmitted as an autosomal recessive trait. The propositus presented with primary amenorrhea and absent thelarche and pubarche. Dynamic tests demonstrated absent spontaneous gonadotropin pulsatility, and no response to either exogenous pulsatile (10 microg/pulse at 90-min intervals over 6 h) or acute (100 microg) GnRH administration. However, she responded to exogenous gonadotropin administration, with a resulting normal pregnancy. Genomic DNA extracted from peripheral blood was PCR amplified using amplimers spanning intron-exon boundaries for the three exons of GnRHR and revealed a homozygous splice junction mutation (G to A transversion) at the intron 1-exon 2 boundary. Her unaffected sister, with a totally normal phenotype, was heterozygous for this mutation. After lymphocyte Epstein-Barr virus transformation, RNA was extracted and subjected to RT-PCR, using primers located in the first and third exons. Results showed a transcript lacking all of exon 2 (exon 2 skipping), with splicing of exon 1 to exon 3. This created a frame shift, generating a coding sequence for three new amino acids, followed by a stop codon. Although it is not clear whether the mutant receptor is actually expressed, the resultant mRNA sequence was presumed to produce a truncated receptor with no binding or signaling capacity.

Adolescent↗

PubMed1999

Three novel small deletion mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia.

The low-density lipoprotein (LDL) receptor gene from 80 unrelated Korean patients with familial hypercholesterolemia (FH) was analyzed to screen for small structural rearrangements that could not be detected by Southern blot hybridization. Three different small deletions were detected in exon 11 of 3 FH patients and were characterized by DNA sequence analysis. Of them two mutations are in-frame 36-bp (FH 2) and 9-bp (FH 34) deletions that result in the loss of twelve amino acids (from Met510 to Ile521) and three amino acids (Thr513, Asp514 and Trp515), respectively. Both mutations are located in the third of the five YWTD motifs of the LDL receptor gene. The third mutation (FH 400) is a 2-bp deletion that shifts the translational reading frame and results in a prematurely terminated receptor protein. The generation of a 36-bp deletion can be explained by the formation of a hairpin-loop structure mediated by inverted repeat sequences. On the other hand, the mechanism responsible for the 9- and the 2-bp deletions is probably strand-slippage mispairing mediated by short direct repeats. All of these three deletions are novel mutations. Each of the three deletions was detected only in a single pedigree out of 80 FH families analyzed.

Adult↗

PubMed1997

Novel "restoration of function" mutagenesis strategy to identify amino acids of the delta-opioid receptor involved in ligand binding.

A novel "restoration of function" mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind delta-selective ligands to an inactive delta/mu receptor chimera in which 10 amino acids of the third extracellular loop of the delta receptor were replaced by the corresponding amino acids from the mu receptor (delta/mu291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for delta-selective ligands. A library of mutants was generated in which some of the 10 amino acids of the mu sequence of delta/mu291-300 were randomly reverted to the corresponding delta amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for delta-selective ligands. Sequence analysis of these revertants revealed that a leucine at position 300, a hydrophobic region (amino acids 295-300), and an arginine at position 291 of the human delta-opioid receptor were present in all revertants. Single and double point mutations were then introduced in delta/mu291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of delta-selective ligands. An increased affinity for delta-selective ligands was observed when the tryptophan 300 (mu residue) of delta/mu291-300 was reverted to a leucine (delta residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of delta-selective ligands to their docking site.

Amino Acid Sequence↗

PubMed1997

Survey and molecular genetics of SHV beta-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12.

Sixty isolates of Enterobacteriaceae resistant to beta-lactam antibiotics were collected over a period of 2 years in Switzerland and screened by hybridization for the carriage of SHV genes. Thirty-four positive strains were found, and their SHV genes were amplified and sequenced. SHV extended-spectrum beta-lactamases (ESBLs) were found: 13 strains contained SHV-2a, 12 harbored SHV-2, and SHV-5 was found twice. Four strains were shown to contain SHV-1. In addition, we report two new SHV variants, termed SHV-11 (non-ESBL) and SHV-12 (ESBL). In spite of the carriage of SHV ESBLs, many strains showed only low resistance to one or more third-generation cephalosporins. In addition, 26 did not transfer the blaSHV gene in mating experiments.

Anti-Bacterial Agents↗

PubMed1993

Small introns in a hepatic cDNA encoding a new glucagon-like peptide 1-type receptor.

A cDNA fragment was PCR amplified at low-stringency from rat DNA, using two degenerated primers corresponding to consensus sequences in the second and third transmembrane segments of members of the secretin receptor family. A synthetic oligodeoxynucleotide based on the 3 end of this fragment, when used in Northern-blot analysis, hybridized to a broad 1.5-3.0 kb band in rat liver. It was then used as a probe for screening a cDNA library generated from size-fractionated rat liver poly(A)+ RNA. Overlapping alignment of sequences from 3 clones identified a 1455 residue open reading frame encoding a 485 amino acid peptide showing 7 putative transmembrane segments and 42% identity with the glucagon-like peptide 1 (GLP-1) receptor. In addition, four small (85-300 bp) introns were variously represented in the isolated clones.

Amino Acid Sequence↗

PubMed1997

Site-directed mutagenesis of the yeast V-ATPase A subunit.

To investigate the function of residues at the catalytic nucleotide binding site of the V-ATPase, we have carried out site-directed mutagenesis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast. Of the three cysteine residues that are conserved in all A subunits sequenced thus far, two (Cys284 and Cys539) appear essential for correct folding or stability of the A subunit. Mutation of the third cysteine (Cys261), located in the glycine-rich loop, to valine, generated an enzyme that was fully active but resistant to inhibition by N-ethylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation. To test the role of disulfide bond formation in regulation of vacuolar acidification in vivo, we have also determined the effect of the C261V mutant on targeting and processing of the soluble vacuolar protein carboxypeptidase Y. No difference in carboxypeptidase Y targeting or processing is observed between the wild type and C261V mutant, suggesting that disulfide bond formation in the V-ATPase A subunit is not essential for controlling vacuolar acidification in the Golgi. In addition, fluid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in the C261V and wild type cells. Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of V-ATPase activity. The E286Q mutant showed labeling by 2-azido-[32P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.

4-Chloro-7-nitrobenzofurazan↗