Cys(577) is a conformationally mobile residue in the ATP-binding domain of the Na,K-ATPase alpha-subunit.

2-[4'-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K(+) (<1 mM); the protective effect K(+) is reversed at higher concentrations. This biphasic effect was also observed with K(+) congeners. In contrast, Na(+) ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of approximately 5 kDa. N-terminal amino acid sequencing identified the peptide (V(545)LGFCH...). This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.

Structural changes associated with the coupling of ATP hydrolysis and cation transport by the Na pump.

Most of the residues associated with cation coordination seem to reside within transmembrane segments of the alpha-subunit of the Na,K-ATPase, whereas amino acids which appear to be involved in the coordination of ATP are found in the major cytoplasmic loop between transmembrane segments M4 and M5 (see Lingrel & Kuntzweiler, 1994; Lutsenko & Kaplan, 1995). The coupling of the two functions of cation transport and ATP hydrolysis involved in the active transport of Na and K ions must involve interactions between these two structural units. This paper summarizes recent experimental results and conclusions of studies on the renal Na,K-ATPase which have employed controlled proteolysis in the presence of physiological ligands, chemical modification with a range of reagents and a variety of functional assays. The data provide evidence for movements between specific transmembrane segments associated with cation-binding conformations and coupled changes which take place in the ATP binding domain. The binding of different cations in the cation-binding domain is sensed in the ATP binding domain and manifested as a change in reactivity. This occurs at amino acid residues which are widely spaced in primary structure. It is apparent that structural changes are transmitted through much of the ATP-binding domain as a consequence of the occupancy of the cation-binding domain. We also provide evidence that both the number and identity of cations bound are also sensed in the ATP-binding domain.

Cloning and molecular characterization of CnTEF1 which encodes translation elongation factor 1alpha in Cryptococcus neoformans.

Degenerate PCR primers were synthesized based upon known translation factor 1alpha (TEF1) sequences. Touchdown PCR with these primers utilizing Cryptococcus neoformans strain M1-106 genomic DNA as template produced a DNA fragment containing a portion of CnTEF1. This DNA fragment was used as a hybridization probe to clone a cDNA version of CnTEF1 from C. neoformans strain B3501. Comparison of the genomic and cDNA nucleotide sequences revealed the presence of six introns in CnTEF1. The nucleotide sequence of CnTEF1 from these two strains of C. neoformans were 98% identical. Codon bias for most amino acids encoded by CnTEF1 was similar to that observed in Saccharomyces cerevisiae for highly expressed genes. This codon bias was also observed in the C. neoformans ACT gene. CnTEF1 encoded a protein (CnEF-1alpha) consisting of 459 amino acids with a calculated MW of 50.3 kDa from C. neoformans strain B3501. CnTEF1 from strain M1-106 encoded a protein with one additional aa. Both C. neoformans proteins possessed a high degree of identity throughout their length to fungal, human, and plant EF-1alpha proteins.

Cloning and characterization of CneMDR1: a Cryptococcus neoformans gene encoding a protein related to multidrug resistance proteins.

CneMDR1, a gene encoding a protein related to several eukaryotic multidrug resistance (MDR) proteins, was identified, cloned, and characterized from a clinical isolate of Cryptococcus neoformans (Cn) (strain M1-106). Polymerase chain reaction (PCR) amplification of a DNA region encompassing conserved motifs of other MDR-like proteins was initially used to identify and clone CneMDR1. Analysis of the corresponding cDNA revealed an open reading frame punctuated by 16 introns. CneMDR1 encoded a protein (CNEMDR1) containing 1408 amino acids (aa) with a predicted mass of approximately 152kDa. Protein structure predictions suggested the presence of two putative 6-transmembrane (TM) domains as well as two ATP-binding domains, structural characteristics typical of ATP-binding cassette (ABC) proteins. Members of this superfamily, which include MDR proteins, are frequently involved in active transport of a variety of substrates across the cell membrane. Pulsed-field gel electrophoresis revealed the presence of 12 chromosomal bands in this clinical isolate of Cn. CneMDR1 was detected by hybridization on chromosome IV. High-stringency hybridization detected only one MDR-like gene. However, a second MDR-like gene (CneMDR2) was discovered during reverse transcriptase-PCR (RT-PCR) amplification using cDNA.

Integrative and replicative genetic transformation of Aureobasidium pullulans.

A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1-alpha (TEF1) adjacent to the 5' end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmBR) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41bp of homologous DNA has also been shown to transform A. pullulans to HmBR. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmBR transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7-8kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmBR A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4kb) was unable to transform A. pullulans to HmBR.

Cloning and characterization of the gene encoding translation elongation factor 1 alpha from Aureobasidium pullulans.

The gene (TEF1) encoding translation elongation factor 1 alpha from the dimorphic fungus Aureobasidium pullulans (Ap) strain R106 has been cloned using Candida albicans TEF1 as a heterologous hybridization probe. Electrophoretic molecular karyotype/hybridization analysis of Ap revealed eight chromosomal bands and suggested that TEF1 resides on chromosome VI. Comparison of the genomic DNA sequence with the cDNA sequence of TEF1 verified the presence of three introns, the first one occurring five nucleotides from the start of translation. The deduced amino acid (aa) sequence encoded a protein consisting of 459 aa (49,663 Da) possessing high identity to a variety of TEF1-encoded proteins. A strong codon bias, similar to that observed in highly expressed yeast genes, was evident in A. pullulans TEF1. The ApTEF1 promoter region showed some sequence similarity to the well-studied TEF1 promoter from Saccharomyces cerevisiae, including a region from -23 to -11. This region exhibited high homology to a promoter upstream activating sequence (UAS) in S. cerevisiae. Such sequences have been shown to be essential promoter elements in genes coding for the highly expressed components of the yeast translation apparatus.

An efficient expression and secretion system based on Bacillus subtilis phage phi 105 and its use for the production of B. cereus beta-lactamase I.

A novel expression system based on the Bacillus subtilis bacteriophage phi 105 has been developed to permit the high-level synthesis and secretion of beta-lactamase I (BlaI) from Bacillus cereus. Shotgun insertion of a promoterless lacZ gene into the phage genome permitted the identification of a clone producing large amounts of beta-galactosidase (beta Gal), indicating the transcription of the reporter gene from a strong phage promoter. The insertion also blocked lysis of the host cell. Although the insertion in the original prophage was complex, plasmid vectors and prophage derivatives have been developed to facilitate the replacement of lacZ with other genes for expression. The new prophages contain two additional mutations: an ind mutation, which greatly enhances the normally poor transformability of phi 105 lysogens, and a cts mutation, which allows thermo-induction of phage development and protein production. Induction of a derivative prophage containing the blaI gene from B. cereus resulted in the production of up to 500 micrograms of secreted BlaI per ml of culture supernatant.

Site-directed mutagenesis and substrate-induced inactivation of beta-lactamase I.

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.