Transport of long chain fatty acids in Escherichia coli. Identification of a membrane protein associated with the fadL gene.

The fadL gene is required for the transport of long chain fatty acids in Escherichia coli (Maloy, S. R., Ginsburgh, C. L., Simons, R. W., and Nunn, W. D. (1981) J. Biol Chem. 256, 3735-3742). In an effort to define the fadL gene product(s), the membrane proteins of 16 independently isolated fadL mutants and wild type strains were compared by two-dimensional gel electrophoretic analysis. These studies indicated that (i) whenever a fadL mutation was present, a 33,000-dalton inner membrane protein was consistently absent, (ii) genetic restoration of mutant alleles to fadL+ resulted in the reappearance of this 33-kDa protein, and (iii) a reversion event mapping to the fadL gene produced a 33-kDa protein with altered electrophoretic properties. Mutations in the other known fad structural genes do not affect 33-kDa protein synthesis and the expression of the 33-kDa protein is physiologically regulated in a manner similar to the known fad enzymes. Overall, these studies suggest there is a close relationship between the 33-kDa inner membrane protein and the fadL gene which putatively codes for a fatty acid transport component(s).

Isolation and genetic characterization of Escherichia coli mutants defective in propionate metabolism.

Escherichia coli mutants defective in propionate metabolism (Prp-) were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Prp- mutants demonstrate a phenotypic inability to grow on odd-chain-length fatty acids. The new genetic locus for the Prp- phenotype maps at approximately 98 min on the E. coli chromosome.

Transport of long and medium chain fatty acids by Escherichia coli K12.

Kinetic, metabolic, and physical parameters of long and medium chain fatty acid transport by Escherichia coli K12 were determined. Uptake of long chain fatty acids (C11-C18:1) mediated by the fadL gene involves concentrative transport. Evidence for this is as follows: (i) characteristic Ki and Vmax values were obtained for long chain fatty acids, (ii) long chain fatty acid transport was inhibited by energy inhibitors, (iii) long chain fatty acids were concentrated 10-fold inside the cell against a concentration gradient, (iv) efflux of transported long chain fatty acids did not occur, and (v) an energy of activation of 11.72 kcal mol-1 and Q10 of 2.3 were obtained for long chain fatty acid transport. The fadL gene product shows some activity with medium chain fatty acids (C7-C10) as well. Medium chain fatty acids also appear to enter the cell by simple diffusion since: (i) medium chain fatty acid transport by fadL strains is not saturable under our assay conditions, (ii) fadL strains do not concentrate medium chain fatty acids against a concentration gradient, and (iii) medium chain fatty acids are available for efflux in fadL strains. Physical parameters of long and medium chain fatty acid transport are also reported. These results present evidence for separate mechanisms of long and medium chain fatty acid transport in E. coli.

Independent localization and regulation of carbamyl phosphate synthetase A polypeptides of Neurospora crassa.

Carbamyl phosphate synthetase A is a two-polypeptide, mitochondrial enzyme of arginine synthesis in Neurospora. The large subunit is encoded in the arg-3 locus and can catalyze formation of carbamyl-P with ammonia as the N donor. The small subunit is encoded in the unlinked arg-2 locus and imparts to the holoenzyme the ability to use glutamine, the biological substrate, as the N donor. By using nonsense mutations of arg-3, it was shown that the small subunit of the enzyme enters the mitochrondrion independently and is regulated in the same manner as it is in wild type. Similarly, arg-2 mutations, affecting the small subunit, have no effect on the localization or the regulation of the large subunit. The two subunits are regulated differently. Like most polypeptides of the pathway, the large subunit is not repressible and derepresses 3- to 5-fold upon arginine-starvation of mycelia. In contrast, the glutamine-dependent activity of the holoenzyme is fully repressible and has a range of variation of over 100-fold. In keeping with this behavior, it is shown here that the small polypeptide, as visualized on two-dimensional gels, is also fully repressible. We conclude that the two subunits of the enzyme are localized independently, controlled independently and over different ranges, and that aggregation kinetics cannot alone explain the unusual regulatory amplitude of the native, two-subunit enzyme. The small subunit molecular weight was shown to be approximately 45,000.

Sources of subcortical projections to the superior colliculus in the cat.

A comprehensive search for subcortical projections to the cat superior colliculus was conducted using the retrograde horseradish peroxidase (HRP) method. Over 40 different subcortical structures project to the superior colliculus. The more notable among these are grouped under the following categories. Visual structures: ventral lateral geniculate nucleus, parabigeminal nucleus, pretectal area (nucleus of the optic tract, posterior pretectal nucleus, nuclei of the posterior commissure). Auditory structures: inferior colliculus (external and pericentral nuclei), dorsomedial periolivary nucleus, nuclei of the trapezoid body, ventral nucleus of the lateral lemniscus. Somatosensory structures: sensory trigeminal complex (all divisions, but mainly the gamma division of nucleus oralis), dorsal column nuclei (mostly cuneate nucleus), and the lateral cervical nucleus. Catecholamine nuclei: locus coeruleus, raphe dorsalis, and the parabrachial nuclei. Cerebellum: medial, interposed, and lateral nuclei, and the perihypoglossal nuclei. Reticular areas: zona incerta, substantia nigra, midbrain tegmentum, nucleus paragigantocellularis lateralis, and the hypothalamus. Evidence is presented that only the parabigeminal nucleus, the nucleus of the optic tract, and the posterior pretectal nucleus project to the superficial collicular layers (striatum griseum superficiale and stratum opticum), while all other afferents terminate in the deeper layers of the colliculus. Also presented is information concerning the rostrocaudal distribution of some of these afferent connections. These findings stress the multiplicity and diversity of inputs to the deeper collicular layers, and more specifically, identify multiple sources of the physiologically well-known representations of the somatic and auditory modalities in the colliculus.