Flexible programs for the prediction of average amphipathicity of multiply aligned homologous proteins: application to integral membrane transport proteins.

Simple flexible programs (TREEMOMENT and PILEUPMOMENT) are described for depicting the average amphipathicity (hydrophobic moment) along multiply aligned sequences of a family of evolutionarily related proteins. The programs are applicable to any number of aligned sequences and can be set for any desired angle corresponding to a residue repeat unit in a protein secondary structural element such as 100 degrees per residue for an alpha-helix or 180 degrees per residue for a beta-strand. These programs can be used to identify amphipathic regions common to the members of a protein family. The use of these programs is exemplified by showing that some families of integral membrane transport proteins (i.e. permeases of the bacterial phosphotransferase system (PTS) and the anion exchangers of animals) exhibit strikingly amphipathic alpha-helical structures immediately preceding the first hydrophobic transmembrane segment of their membrane-embedded domain(s). Other families, such as the major facilitator superfamily of uniporters, symporters and antiporters, do not exhibit this structural feature. The amphipathic structures in PTS permeases have been implicated in membrane insertion during biogenesis.

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.

The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins.

A previous report identified and classified a small family of gram-negative bacterial drug and heavy metal efflux permeases, now commonly referred to as the RND family (TC no. 2.6). We here show that this family is actually a ubiquitous superfamily with representation in all major kingdoms. We report phylogenetic analyses that define seven families within the RND superfamily as follows: (1) the heavy metal efflux (HME) family (gram negative bacteria), (2) the hydrophobe/amphiphile efflux-1 (HAE1) family (gram negative bacteria), (3) the nodulation factor exporter (NFE) family (gram negative bacteria), (4) the SecDF protein-secretion accessory protein (SecDF) family (gram negative and gram positive bacteria as well as archaea), (5) the hydrophobe/amphiphile efflux-2 (HAE2) family (gram positive bacteria), (6) the eukaryotic sterol homeostasis (ESH) family, and (7) the hydrophobe/amphiphile efflux-3 (HAE3) family (archaea and spirochetes). Functionally uncharacterized proteins were identified that are members of the RND superfamily but fall outside of these seven families. Some of the eukaryotic homologues function as enzymes and receptors instead of (or in addition to) transporters. The sizes and topological patterns exhibited by members of all seven families are shown to be strikingly similar, and statistical analyses establish common descent. Multiple alignments of proteins within each family allow derivation of family-specific signature sequences. Structural, functional, mechanistic and evolutionary implication of the reported results are discussed.

The major facilitator superfamily.

In 1998 we updated earlier descriptions of the largest family of secondary transport carriers found in living organisms, the major facilitator superfamily (MFS). Seventeen families of transport proteins were shown to comprise this superfamily. We here report expansion of the MFS to include 29 established families as well as five probable families. Structural, functional, and mechanistic features of the constituent permeases are described, and each newly identified family is shown to exhibit specificity for a single class of substrates. Phylogenetic analyses define the evolutionary relationships of the members of each family to each other, and multiple alignments allow definition of family-specific signature sequences as well as all well-conserved sequence motifs. The work described serves to update previous publications and allows extrapolation of structural, functional and mechanistic information obtained with any one member of the superfamily to other members with limitations determined by the degrees of sequence divergence.

Sequence analysis and expression of the filamentous phage phi Lf gene I encoding a 48-kDa protein associated with host cell membrane.

One viral strand of phi Lf, a filamentous phage of Xanthomonas campestris pv.campestris, the open reading frame (ORF440) behind gene VI was identified as gene I. This gene codes for pI protein (440 aa, 48 kDa) which was shown to be membrane-bound in the phi Lf-infected host cell by Western blot analysis using the antibody raised against the protein expressed in Escherichia coli. Its predicted amino acid sequence has a nucleotide-binding motif in the N-terminal 97 aa and a membrane-spanning domain (aa 221 to 236). These structural features are characteristic of pIs of several filamentous phages which are transmembrane proteins required for phage assembly. Thus far, nine phi Lf genes have been identified which are organized in the order GII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI, similar to the genome organization of E. coli filamentous phages.

Identification of gene VI of filamentous phage phi Lf coding for a 10-kDa minor coat protein.

ORF95 in the filamentous phage phi Lf genome, locating behind gIII, was identified to be the gene (gVI) coding for minor coat protein pVI (95 amino acids, 10,245 dal). It was shown to be virion associated by Western blot analysis of chloroform-treated phage particles. Computer analysis predicted two transmembrane regions for this protein. Since no signal peptide was suggested and the size estimated by SDS-polyacrylamide gel electrophoresis matches that deduced from nucleotide sequence, it appears to be incorporated into the phage particle as its primary translational product. After completion of this study, eight genes organizing into an order of gVII-gX-gV-gVII-gIX-gIII-gIII-gVI have been identified for phi Lf.

The development and testing of a prototype on-line radioiodine monitor for nuclear power stations.

A prototype on-line monitor has been developed which is capable of detecting radioiodine in the presence of as much as 1 X 10(6) higher concentration of noble gases. The system contains two identical radiation monitoring chambers through which the monitored air and a purging gas alternately cycle. Each chamber contains a silver zeolite filter which has a high retention of the various forms of airborne radioiodine but low retention of noble gases. During the purging cycle the radioactive noble gases are quickly purged from the filter and chamber and the lower levels of radioiodine accumulated on the filter are detected. This system has been successfully tested using short-lived radionuclides simulating vented reactor gases resulting from an abnormal condition.