Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium.

An epithelial cell line from pig kidney (LLC-PK1) with properties of proximal tubular cells can be maintained indefinitely in hormone-supplemented serum-free medium. Continuous growth requires the presence of seven factors: transferrin, insulin, selenium, hydrocortisone, triiodothyronine, vasopressin, and cholesterol. The hormone-defined medium (a) supports growth of LLC-PK1 cells at a rate of approaching that observed in serum-supplemented medium; (b) allows vectorial transepithelial salt and fluid transport as measured by hemicyst formation; and (c) influences cell morphology. The vasopressin dependency for growth and morphology can be partially replaced by isobutylmethylxanthine or dibutyryl cyclic AMP. The medium has been used to isolate rabbit proximal tubular kidney epithelial cells free of fibroblasts.

Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK).

Madin-Darby canine kidney (MDCK) cells grown in tissue culture have the morphological properties of distal tubular epithelial cells, form tight junctions, and lack several proximal tubular enzyme markers. Adenylate cyclase in these cells was stimulated by vasopressin, oxytocin, prostaglandins E1 and E2, glucagon, and cholera toxin. Hormone-stimulated adenylate cyclase activity in isolated membrane preparations was dependent on low concentrations of GTP and had the MgCl2 and pH optima expected for the kidney enzyme. The results, as well as the demonstration of enhanced hemicyst formation induced by cyclic AMP, suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cell of origin. When MDCK cells were injected into baby nude mice, continuous nodule growth was observed until adulthood was attained. Histological studies revealed the presence of two cell types: normal mouse fibroblasts which comprise 80--90% of the solid nodule mass, and MDCK cells, which formed epithelial sheets lining internal fluid-filled glands. Electron microscope analysis showed that the mucosal surfaces of the cells were characterized by microvilli which faced the lumen of the glands, that adjacent MDCK cells were joined by tight junctions, and that the serosal surfaces of the epithelial sheets were characterized by smooth plasma membranes which were lined by a continuous basement membrane. These observations lead to the conclusion that the MDCK cells retain regional differentiation of their plasma membranes and the ability to regenerate kidney tubule-like structures in vivo.

Transport proteins in bacteria: common themes in their design.

Bacterial transport proteins mediate passive and active transport of small solutes across membranes. Comparison of amino acid sequences shows strong conservation not only among bacterial transporters, but also between them and many transporters of animal cells; thus the study of bacterial transporters is expected to contribute to our understanding of transporters in more complex cells. During the last few years, structures of three bacterial outer membrane transporters were solved by x-ray crystallography. Much progress has also occurred in the biochemical and molecular genetic studies of transporters in the cytoplasmic membranes of bacteria, and a unifying design among membrane transporters is gradually emerging. Common structural motives and evolutionary origins among transporters with diverse energy-coupling mechanisms suggest that many transporters contain a central module forming a transmembrane channel through which the solute may pass. Energy-coupling mechanisms can be viewed as secondary features added on to these fundamental translocation units.

A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport.

Many transport proteins of bacteria and eukaryotes are thought to possess a common structural motif of 12 transmembrane-spanning alpha-helical segments. In this report we use statistical methods to establish that five families or clusters of these facilitators comprise a single superfamily. The five clusters include: (1) drug-resistance proteins, (2) sugar facilitators, (3) facilitators for Krebs cycle intermediates, (4) phosphate ester-phosphate antiporters and (5) a distinct group of oligosaccharide-H+ symporters. Over 50 transporters of bacteria, lower eukaryotes, plants and animals, and one putative bacterial transcriptional regulatory protein are members of this superfamily, which we term the 'major facilitator superfamily' (MFS).

Salmonella virulence: new clues to intramacrophage survival.

Salmonella are capable of survival in macrophages (cells which have evolved specific mechanisms to kill pathogenic bacteria). One mechanism involves the bactericidal peptides called defensins which insert into phospholipid bilayers to generate transmembrane pores. Synthesis of the Salmonella gene products which determine resistance to defensins has been found to be under the control of a transcriptional regulatory protein termed PhoP.

Regulation of bacterial physiological processes by three types of protein phosphorylating systems.

A single type of protein-phosphorylating system, the ATP-dependent protein kinases, is employed in the regulation of a variety of cellular physiological processes in eukaryotes. By contrast, recent work with bacteria has revealed that three types of protein-phosphorylating systems are involved in regulation: (1) the classical protein kinases, (2) the newly discovered sensor-kinase/response-regulator systems, and (3) the multifaceted phosphoenolpyruvate-dependent phosphotransferase system. Physiological and mechanistic aspects of these three evolutionarily distinct systems are discussed.

Protein phosphorylation and regulation of carbon metabolism in gram-negative versus gram-positive bacteria.

Bacteria impose regulatory mechanisms on metabolic processes to ensure that the needs of the cell are met but not exceeded. Here, we discuss the basic features of a mechanism by which carbohydrate catabolism in Gram-positive bacteria is regulated. Although the physiological consequences of this regulation are the same as in Gram-negative bacteria, the mechanism is entirely different. These regulatory processes evidently evolved late, after the divergence of Gram-negative bacteria, even though the targets of regulation are universal.

A functional-phylogenetic classification system for transmembrane solute transporters.

A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional assignments of newly sequenced transport proteins that will result from future genome sequencing projects.

Major facilitator superfamily.

The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane alpha-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.

Modular multidomain phosphoryl transfer proteins of bacteria.

Recent phylogenetic and structural analyses of multidomain phosphoryl transfer proteins of bacteria have revealed that interdomain (but not intradomain) splicing and fusion, as well as domain duplication and deletion, have occurred frequently during evolution. These events have been found to be exceedingly rare in certain other protein families. Domain-shuffling events are illustrated by examples from the superfamilies of phosphoenolpyruvate-dependent sugar phosphotransferase systems, their transcriptional regulatory protein targets of phosphorylation, sensor autokinase/response regulator signal transduction systems, and permeases of the ATP-binding-cassette type.

Genome archeology leading to the characterization and classification of transport proteins.

In the study of transmembrane transport, molecular phylogeny provides a reliable guide to protein structure, catalytic and noncatalytic transport mechanisms, mode of energy coupling and substrate specificity. It also allows prediction of the evolutionary history of a transporter family, leading to estimations of its age, source, and route of appearance. Phylogenetic analyses, therefore, provide a rational basis for the characterization and classification of transporters. A universal classification system has been described, based on both function and phylogeny, which has been designed to be applicable to all currently recognized and yet-to-be discovered transport proteins found in living organisms on Earth.

The structure of an energy-coupling protein from bacteria, IIBcellobiose, reveals similarity to eukaryotic protein tyrosine phosphatases.

BACKGROUND: . The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates the energy-driven uptake of carbohydrates and their concomitant phosphorylation. In addition, the PTS is intimately involved in the regulation of a variety of metabolic and transcriptional processes in the bacterium. The multiprotein PTS consists of a membrane channel and at least four cytoplasmic proteins or protein domains that sequentially transfer a phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. Determination of the three-dimensional structure of the IIB enzymes within the multiprotein complex would provide insights into the mechanisms by which they promote efficient transport by the membrane channel IIC protein and phosphorylate the transported carbohydrate on the inside of the cell. RESULTS: . The crystal structure of the IIB enzyme specific for cellobiose, IIBcellobiose (molecular weight 11.4 kDa), has been determined to a resolution of 1.8 and refined to an R factor of 18.7% (Rfree of 24. 1%). The enzyme consists of a single four-stranded parallel beta sheet flanked by helices on both sides. The phosphorylation site (Cys 10) is located at the C-terminal end of the first beta strand. No positively charged residues, which could assist in phosphoryl-transfer, can be found in or near the active site. The fold of IIBcellobiose is remarkably similar to that of the mammalian low molecular weight protein tyrosine phosphatases. CONCLUSIONS: . A comparison between IIBcellobiose and the structurally similar low molecular weight protein tyrosine phosphatases provides insight into the mechanism of the phosphoryltransfer reactions in which IIBcellobiose is involved. The differences in tertiary structure and active-site composition between IIBcellobiose and the glucose-specific IIBglucose give a structural explanation why the carbo-hydrate-specific components of different families cannot complement each other.

Relationship of cell growth behavior in vitro to tumorigenicity in athymic nude mice.

The serum requirements, anchorage requirements, saturation densities, and contact inhibition responses of a variety of mammalian cell lines were determined under uniform conditions. The serum requirement of both transformed and normal cells was a sensitive function of initial plating density. Cloning efficiency on irradiated mouse monolayers was found to be an invalid indicator of contact inhibition of growth, since most cell lines that failed to form visible colonies on cell monolayers nonetheless proliferated on these monolayers. When normal and neoplastic cells from a variety of sources were examined, none of the growth parameters that serve to define the transformed state in vitro correlated consistently with cellular tumorigenicity in athymic nude mice. It is concluded that the most reliable and physiologically meaningful assay for malignant transformation is, at present, cellular tumorigenicity in athymic nude mice.

Growth control of heterologous tissue culture cells in the congenitally athymic nude mouse.

A variety of heterologous mammalian cells were inoculated into nude mice and scored for tumorigenicity. The cells tested were from primary cell cultures, established cell lines of neoplastic origin, established cell lines of nontumor origin, and primary cell cultures transformed by oncogenic viruses. Regardless of the animal species of origin, every cell line that was tumorigenic in some other animal host and every cell line of neoplastic origin was tumorigenic in nude mice. Several tissue culture cells lines capable of indefinite growth in vitro failed to form tumors in nude mice, and the basis of this growth suppression was investigated. The findings suggest that the failure of an established cell line to form tumors in nude mice is an authentic response to host-mediated growth-regulatory signals.

The amoebapore superfamily.

Amoebapores, synthesized by human protozoan parasites, form ion channels in target cells and artificial lipid membranes. The major pathogenic effect of these proteins is due to their cytolytic capability which results in target cell death. They comprise a coherent family and are homologous to other proteins and protein domains found in eight families. These families include in addition to the amoebapores (1) the saposins, (2) the NK-lysins and granulysins, (3) the pulmonary surfactant proteins B, (4) the acid sphingomyelinases, (5) acyloxyacyl hydrolases and (6) the aspartic proteases. These amoebapore homologues have many properties in common including membrane binding and stability. We note for the first time that a new protein, countin, from the cellular slime mold, Dictyostelium discoideum, comprises the eighth family within this superfamily. All currently sequenced members of these eight families are identified, and the structural, functional and phylogenetic properties of these proteins are discussed.

A functional superfamily of sodium/solute symporters.

Eleven families of sodium/solute symporters are defined based on their degrees of sequence similarities, and the protein members of these families are characterized in terms of their solute and cation specificities, their sizes, their topological features, their evolutionary relationships, and their relative degrees and regions of sequence conservation. In some cases, particularly where site-specific mutagenesis analyses have provided functional information about specific proteins, multiple alignments of members of the relevant families are presented, and the degrees of conservation of the mutated residues are evaluated. Signature sequences for several of the eleven families are presented to facilitate identification of new members of these families as they become sequenced. Phylogenetic tree construction reveals the evolutionary relationships between members of each family. One of these families is shown to belong to the previously defined major facilitator superfamily. The other ten families do not show sufficient sequence similarity with each other or with other identified transport protein families to establish homology between them. This study serves to clarify structural, functional and evolutionary relationships among eleven distinct families of functionally related transport proteins.

The amino acid/auxin:proton symport permease family.

Amino acids and their derivatives are transported into and out of cells by a variety of permease types which comprise several distinct protein families. We here present a systematic analysis of a group of homologous transport proteins which together comprise the eukaryotic-specific amino acid/auxin permease (AAAP) family (TC #2. 18). In characterizing this family, we have (1) identified all sequenced members of the family, (2) aligned their sequences, (3) identified regions of striking conservation, (4) derived a family-specific signature sequence, and (5) proposed a topological model that appears to be applicable to all members of the family. We have also constructed AAAP family phylogenetic trees and dendrograms using six different programs that allow us to trace the evolutionary history of the family, estimate the relatedness of proteins from dissimilar organismal phyla, and evaluate the reliability of the different programs available for phylogenetic studies. The TREE and neighbor-joining programs gave fully consistent results while CLUSTAL W gave similar but non-identical results. Other programs gave less consistent results. The phylogenetic analyses reveal (1) that many plant AAAP family proteins arose recently by multiple gene duplication events that occurred within a single organism, (2) that some plant members of the family with strikingly different specificities diverged early in evolutionary history, and (3) that AAAP family proteins from fungi and animals diverged from the plant proteins long ago, possibly when animals, plants and fungi diverged from each other. The Neurospora protein nevertheless exhibits overlapping specificity with those found in plants. Preliminary evidence is presented suggesting that proteins of the AAAP family are distantly related to proteins of the large ubiquitous amino acid/polyamine/choline family (TC #2.3) as well as to those of two small bacterial amino acid transporter families, the ArAAP family (TC #2.42) and the STP family (TC #2.43).

Homologues of archaeal rhodopsins in plants, animals and fungi: structural and functional predications for a putative fungal chaperone protein.

The microbial rhodopsins (MR) are homologous to putative chaperone and retinal-binding proteins of fungi. These proteins comprise a coherent family that we have termed the MR family. We have used modeling techniques to predict the structure of one of the putative yeast chaperone proteins, YRO2, based on homology with bacteriorhodopsins (BR). Availability of the structure allowed depiction of conserved residues that are likely to be of functional significance. The results lead us to predict an extracellular protein folding function and a transmembrane proton transport pathway. We suggest that protein folding is energized by a novel mechanism involving the proton motive force. We further show that MR family proteins are distantly related to a family of fungal, animal and plant proteins that include the human lysosomal cystine transporter (LCT) of man (cystinosin), mutations in which cause cystinosis. Sequence and phylogenetic analyses of both the MR family and the LCT family are reported. Proteins in both families are of the same approximate size, exhibit seven putative transmembrane alpha-helical spanners (TMSs) and show limited sequence similarity. We show that the LCT family arose by an internal gene duplication event and that TMSs 1-3 are homologous to TMSs 5-7. Although the same could not be demonstrated statistically for MR family members, homology with the LCT family suggests (but does not prove) a common evolutionary pathway. Thus, TMSs 1-3 and 5-7 in both LCT and MR family members may share a common origin, accounting for their shared structural features.

Evolution of permease diversity and energy-coupling mechanisms with special reference to the bacterial phosphotransferase system.

Different classes of apparently unrelated permeases couple different forms of energy to solute transport. While the energy coupling mechanisms utilized by the different permease classes are clearly distinct, it is proposed, based on structural comparisons, that many of these permeases possess transmembrane, hydrophobic domains which are evolutionarily related. Carriers may have arisen from transmembrane pore-forming proteins, and the protein constituents or domains which are specifically responsible for energy coupling may have had distinct origins. Thus, complex permeases may possess mosaic structures. This suggestion is substantiated by recent findings regarding the evolutionary origins of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). Mechanistic implications of this proposal are presented.

Phylogenetic characterization of novel transport protein families revealed by genome analyses.

As a result of recent genome sequencing projects as well as detailed biochemical, molecular genetic and physiological experimentation on representative transport proteins, we have come to realize that all organisms possess an extensive but limited array of transport protein types that allow the uptake of nutrients and excretion of toxic substances. These proteins fall into phylogenetic families that presumably reflect their evolutionary histories. Some of these families are restricted to a single phylogenetic group of organisms and may have arisen recently in evolutionary time while others are found ubiquitously and may be ancient. In this study we conduct systematic phylogenetic analyses of 26 families of transport systems that either had not been characterized previously or were in need of updating. Among the families analyzed are some that are bacterial-specific, others that are eukaryotic-specific, and others that are ubiquitous. They can function by either a channel-type or a carrier-type mechanism, and in the latter case, they are frequently energized by coupling solute transport to the flux of an ion down its electrochemical gradient. We tabulate the currently sequenced members of the 26 families analyzed, describe the properties of these families, and present partial multiple alignments, signature sequences and phylogenetic trees for them all.