The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding cassette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete loss of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.

No abstract

A novel dynein-related transcript (designated DNEL1) from human adult testis has been identified that can encode a protein with a size of 91087 Da. The complete nucleotide sequence of the open reading frame is the first to be described for a human dynein-related gene. Northern blot analysis of mRNA from 16 different tissues has shown that DNEL1 is expressed specifically in testis. Analysis of somatic cell hybrids has mapped DNEL1 to Chromosome (Chr) 17. Analysis of a panel of 129 whole genome radiaton hybrid clones including 17q22-q25.3 has placed DNEL1 in 17q distal to the ERBA2L locus. DNEL1 shares a high degree of sequence identity and amino acid similarity with the C-terminal region of the outer arm axonemal dynein beta-heavy chains derived from sea urchin and other species, but not to any gene encoding dynein intermediate or light chains described to date. The close similarity of DNEL1 to the C-terminal part of the axonemal beta-heavy chain may suggest an origin from a common progenitor gene and the testis-specific pattern of expression a possible role in sperm development or motility.

No abstract