Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

Identification of a Subpopulation of Marrow MSC-Derived Medullary Adipocytes That Express Osteoclast-Regulating Molecules: Marrow Adipocytes Express Osteoclast Mediators

Increased marrow medullary adipogenesis and an associated decrease in bone mineral density, usually observed in elderly individuals, is a common characteristic in senile osteoporosis. In this study we investigated whether cells of the medullary adipocyte lineage have the potential to directly support the formation of osteoclasts, whose activity in bone leads to bone degradation. An in vitro mesenchymal stem cell (MSC)-derived medullary adipocyte lineage culture model was used to study the expression of the important osteoclast mediators RANKL, M-CSF, SDF-1, and OPG. We further assessed whether adipocytes at a specific developmental stage were capable of supporting osteoclast-like cell formation in culture. In vitro MSC-derived medullary adipocytes showed an mRNA and protein expression profile of M-CSF, RANKL, and OPG that was dependent on its developmental/metabolic stage. Furthermore, RANKL expression was observed in MSC-derived adipocytes that were at a distinct lineage stage and these cells were also capable of supporting osteoclast-like cell formation in co-cultures with peripheral blood mononuclear cells. These results suggest a connection between medullary adipocytes and osteoclast formation in vivo and may have major significance in regards to the mechanisms of decreased bone density in senile osteoporosis.     

Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

Differential In Vivo Regulation of Steroid Hormone Receptor Activation by Cdc37p

The CDC37 gene is essential for the activity of p60^v-src when expressed in yeast cells. Since the activation pathway for p60^v-src and steroid hormone receptors is similar, the present study analyzed the hormone-dependent transactivation by androgen receptors and glucocorticoid receptors in yeast cells expressing a mutant version of the CDC37 gene. In this mutant, hormone-dependent transactivation by androgen receptors was defective at both permissive and restrictive temperatures, although transactivation by glucocorticoid receptors was mildly defective only at the restrictive temperature. Cdc37p appears to function via the androgen receptor ligand-binding domain, although it does not influence receptor hormone-binding affinity. Models for Cdc37p regulation of steroid hormone receptors are discussed.     

Lysosome-related organelles.

Lysosomes are membrane-bound cytoplasmic organelles involved in intracellular protein degradation. They contain an assortment of soluble acid-dependent hydrolases and a set of highly glycosylated integral membrane proteins. Most of the properties of lysosomes are shared with a group of cell type-specific compartments referred to as 'lysosome-related organelles', which include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and Drosophila pigment granules. In addition to lysosomal proteins, these organelles contain cell type-specific components that are responsible for their specialized functions. Abnormalities in both lysosomes and lysosome-related organelles have been observed in human genetic diseases such as the Chediak-Higashi and Hermansky-Pudlak syndromes, further demonstrating the close relationship between these organelles. Identification of genes mutated in these human diseases, as well as in mouse and Drosophila: pigmentation mutants, is beginning to shed light on the molecular machinery involved in the biogenesis of lysosomes and lysosome-related organelles.     

Role of α-Factor and the Mfα1 α-Factor Precursor in Mating in Yeast

The peptide pheromones secreted by a and α cells (called a-factor and α-factor, respectively) are each encoded by two structural genes. For strains of either mating type, addition of exogenous pheromone does not alleviate the mating defect of mutants with disruptions of both structural genes. In addition, a particular insertion mutation in the major α-factor structural gene (MFα1) that should result in an altered product inhibits α mating. These results suggested that the pheromone precursors (the MFα1 pro region in particular) might play a second role in mating separate from the role of pheromone production. To analyze the role of α-factor and the MFα1 precursor in α mating, we have constructed two classes of mutants. The mating defects of mutants that should produce the MFα1 pro region peptide but no α-factor could not be alleviated by addition of exogenous α-factor in crosses to a wild-type a strain, indicating that the previous results were not due to an inability of the disruption mutants to produce the pro region peptide. Mutants able to produce α-factor, but with a variety of alterations in MFα1 precursor structure, mated at levels proportional to the levels of α-factor produced, suggesting that the only role of the α-factor precursor in mating is to produce α-factor. Both of these results argue against a role for the MFα1 pro region separate from its role in α-factor production. We also describe results that show that in vivo production of α-factor'' (the form of α-factor encoded by one of the two α-factor repeats of MFα2) is equivalent to the major form of α-factor for induction of all responses necessary for mating. We discuss the implications of these results on the role of the pheromones in mating.     

Ultrastructural characterization of embryonic chick cartilage proteoglycan core protein and the mapping of a monoclonal antibody epitope.

The ultrastructure of embryonic chick cartilage proteoglycan core protein was investigated by electron microscopy of specimens prepared by low angle shadowing. The molecular images demonstrated a morphological substructural arrangement of three globular and two linear regions within each core protein. The internal globular region (G2) was separated from two terminally located globular regions (G1 and G3) by two elongated strands with lengths of 21 +/- 3 nm (E1) and 105 +/- 22 nm (E2). The two N-terminal globular regions, separated by the 21-nm segment, were consistently visualized in well spread molecules and showed little variation in the length of the linear segment connecting them. The E2 segment, however, was quite variable in length, and the C-terminal globular region (G3) was detected in only 53% of the molecules. The G1, G2, and G3 regions in chick core protein were 10.1 +/- 1.7 nm, 9.7 +/- 1.3 nm, and 8.3 +/- 1.3 nm in diameter, respectively. These results are similar to those described previously for proteoglycan core proteins isolated from rat chondrosarcoma, bovine nasal cartilage, and pig laryngeal cartilage (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). However, a significant difference was detected between the length of the elongated strand (E2) of core proteins isolated from chick cartilage, E2 length = 105 +/- 22 nm, compared to bovine nasal cartilage, E2 length = 260 +/- 39 nm. The epitope of the proteoglycan core protein-specific monoclonal antibody, S103L, was visualized by electron microscopy, and the distance from the core protein N terminus to the S103L binding site was measured. The S103L binding site was localized to the E2 region, 111 +/- 20 nm from the G1 (N terminus) domain and 34 nm from the G3 (C terminus) domain. cDNA clones selected from an expression vector library of chicken cartilage mRNA also show this epitope to be located near the C-terminal region (R. C. Krueger, T. A. Fields, J. Mensch, and B. Schwartz (1990) J. Biol. Chem. 265, 12088-12097).