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Dystroglycans are essential transmembrane adhesion receptors for laminin. α-Dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of β-dystroglycan. β-Dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin–glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of β-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells.

The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases; the sequences of ovine CPT1A and CPT1B cDNAs have the accession numbers Y18387 and AJ272435 respectively and the partial adipose tissue and liver CPT1A clones have the accession numbers Y18830 and Y18829 respectively. Fatty acid and ketone body metabolism differ considerably between monogastric and ruminant species. The regulation of the key enzymes involved may differ accordingly. Carnitine palmitoyltransferase 1 (CPT 1) is the key locus for the control of long-chain fatty acid beta-oxidation and liver ketogenesis. Previously we showed that CPT 1 kinetics in sheep and rat liver mitochondria differ. We cloned cDNAs for both isoforms [liver- (L-) and muscle- (M-)] of ovine CPT 1 in order to elucidate the structural features of these proteins and their genes ( CPT1A and CPT1B ). Their deduced amino acid sequences show a high degree of conservation compared with orthologues from other mammalian species, with the notable exception of the N-terminus of ovine M-CPT 1. These differences were also present in bovine M-CPT 1, whose N-terminal sequence we determined. In addition, the 5'-end of the sheep CPT1B cDNA suggested a different promoter architecture when compared with previously characterized CPT1B genes. Northern blotting revealed differences in tissue distribution for both CPT1A and CPT1B transcripts compared with other species. In particular, ovine CPT1B mRNA was less tissue restricted, and the predominant transcript in the pancreas was CPT1B. Expression in yeast allowed kinetic characterization of the two native enzymes, and of a chimaera in which the distinctive N-terminal segment of ovine M-CPT 1 was replaced with that from rat M-CPT 1. The ovine N-terminal segment influences the kinetics of the enzyme for both its substrates, such that the K (m) for palmitoyl-CoA is decreased and that for carnitine is increased for the chimaera, relative to the parental ovine M-CPT 1.

Using expressed sequence tag data, we obtained a cDNA for a carnitine palmitoyltransferase I (CPT I)-like molecule from Drosophila melanogaster. The cDNA encodes a 782-residue protein that shows 49% and 48% sequence identity with the rat liver and skeletal-muscle isoforms of CPT I respectively. The sequence has two predicted membrane-spanning regions, suggesting that it adopts the same topology as its mammalian counterparts. The sequence contains all the residues that have been shown to be characteristic of carnitine acetyltransferases. Expression in the yeast Pichia pastoris confirmed that the cDNA does encode a CPT enzyme. The activity was found to be associated with a mitochondria-enriched fraction. Kinetic analysis revealed a K(m) for carnitine of 406 microM and a K(m) for palmitoyl-CoA of 105 microM. The CPT activity was very sensitive to inhibition by malonyl-CoA, with an IC(50) of 0.74 microM when the activity was assayed with 35 microM palmitoyl-CoA and 1% (w/v) albumin at pH 7.0. A histidine residue at position 140 in rat liver CPT I has been indicated to be important for inhibition by malonyl-CoA. The equivalent residue (position 136) in Drosophila CPT I is arginine, implying that any basic residue might be compatible with such sensitivity. Evidence is presented that, unlike in mammals, Drosophila has only a single CPT I gene. Sequences suggesting the existence of a splice variant in the 5' untranslated region were found; this was consistent with the existence of two promoters for the CPT I gene.