The yeast mitochondrial transport proteins: new sequences and consensus residues, lack of direct relation between consensus residues and transmembrane helices, expression patterns of the transport protein genes, and protein-protein interactions with other proteins

Mitochondrial transport proteins (MTP) typically are homodimeric with a 30-kDa subunit with six transmembrane helices. The subunit possesses a sequence motif highly similar to Pro X Asp/Glu X X Lys/Arg X Arg within each of its three similar 10-kDa segments. Four (YNL083W, YFR045W, YPR021C, YDR470C) of the 35 yeast (S. cerevisiae) MTP genes were resequenced since the masses of their proteins deviate significantly from the typical 30 kDa. We now find these four proteins to have 545, 285, 902, and 502 residues, respectively. Together with only four other MTPs, the sequences of YPR021C and YDR470C show substitutions of some of the five residues that are absolutely conserved among the 12 MTPs with identified transport function and 17 other MTPs. We do now find these five consensus residues also in the new sequences of YNL083W and YFR045W. Additional analyses of the 35 yeast MTPs show that the location of transmembrane helix sequences do not correlate with the general consensus residues of the MTP family; protein segments connecting the six transmembrane helices and facing the intermembrane space are not uniformly short (about 20 residues) or long (about 40 residues) when facing the matrix; most MTPs have at least one transmembrane helix for which the sum of the negative hydropathy values of all residues yields a very small negative value, suggesting a membrane location bordering polar faces of other transmembrane helices or a non-transmembrane location. The extra residues of the three large MTPs are hydrophilic and at the N-terminal. The 200-residue N-terminal segment of YNL083W has four putative Ca2+-binding sites. The 500-residue N-terminal segment of YPR021C shows sequence similarity to enzymes of nucleic acid metabolism. cDNA microarray data show that YNL083W is expressed solely during sporulation, while the expressions of YFR045W, YPR021C, and YDR470C are induced by various stress situations. These results also show that the 35 MTP genes are expressed under a rather diverse set of metabolic conditions that may help identify the function of the proteins. Interestingly, yeast two-hybrid screens, that will also be useful in identifying the function of MTPs, indicate that MIR1, AAC3, YOR100C, and YPR011C do interact with non-MTPs.     

Evolution of the MIP family of integral membrane transport proteins

Six integral membrane proteins of bacterial, animal, and plant origin, which are believed to function in solute transport, share sequence identity and are grouped together as members of the MIP family. These include the Escherichia coli glycerol facilitator, the major intrinsic protein from bovine lens fibre junction membranes, a plant tonoplast membrane protein, a soybean protein from the peribacteroid membrane, and a Drosophila neurogenic protein. These proteins, each of which appears to consist of six transmembrane helical segments per subunit, apparently arose by internal duplication of a three-transmembrane segment. Phylogenetic‘trees’interrelating these proteins and segments are presented.     

Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins

The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.     

Phylogeny and evolution of the major intrinsic protein family

BACKGROUND INFORMATION: MIPs (major intrinsic proteins) form channels across biological membranes that control recruitment of water and small solutes such as glycerol and urea in all living organisms. Because of their widespread occurrence and large number, MIPs are a sound model system to understand evolutionary mechanisms underlying the generation of protein structural and functional diversity. With the recent increase in genomic projects, there is a considerable increase in the quantity and taxonomic range of MIPs in molecular databases. RESULTS: In the present study, I compiled more than 450 non-redundant amino acid sequences of MIPs from NCBI databases. Phylogenetic analyses using Bayesian inference reconstructed a statistically robust tree that allowed the classification of members of the family into two main evolutionary groups, the GLPs (glycerol-uptake facilitators or aquaglyceroporins) and the water transport channels or AQPs (aquaporins). Separate phylogenetic analyses of each of the MIP subfamilies were performed to determine the main groups of orthology. In addition, comparative sequence analyses were conducted to identify conserved signatures in the MIP molecule. CONCLUSIONS: The earliest and major gene duplication event in the history of the MIP family led to its main functional split into GLPs and AQPs. GLPs show typically one single copy in microbes (eubacteria, archaea and fungi), up to four paralogues in vertebrates and they are absent from plants. AQPs are usually single in microbes and show their greatest numbers and diversity in angiosperms and vertebrates. Functional recruitment of NOD26-like intrinsic proteins to glycerol transport due to the absence of GLPs in plants was highly supported. Acquisition of other MIP functions such as permeability to ammonia, arsenite or CO2 is restricted to particular MIP paralogues. Up to eight fairly conserved boxes were inferred in the primary sequence of the MIP molecule. All of them mapped on to one side of the channel except the conserved glycine residues from helices 2 and 5 that were found in the opposite side.     

Phylogenetic Characterization of the MIP Family of Transmembrane Channel Proteins

The ubiquitous major intrinsic protein (MIP) family includes several transmembrane channel proteins known to exhibit specificity for water and/or neutral solutes. We have identified 84 fully or partially sequenced members of this family, have multiply aligned over 50 representative, divergent, fully sequenced members, have used the resultant multiple alignment to derive current MIP family-specific signature sequences, and have constructed a phylogenetic tree. The tree reveals novel features relevant to the evolutionary history of this protein family. These features plus an evaluation of functional studies lead to the postulates: (i) that all current MIP family proteins derived from two divergent bacterial paralogues, one a glycerol facilitator, the other an aquaporin, and (ii) that most or all current members of the family have retained these or closely related physiological functions. Received: 19 April 1996/Revised: 3 June 1996     

水稻与拟南芥跨膜蛋白14家族的分子进化特征与功能分析

脂类既是植物生命活动重要的能量来源,也是细胞膜系统不可或缺的结构成分,在植物生长发育和逆境反应等生命活动过程中都起到至关重要的作用。随着脂类代谢研究的不断深入,植物脂类合成通路已渐渐明晰,其中连通不同细胞器间脂类合成中间物质运送的膜蛋白也正被不断发现,但对质体脂类转运蛋白还鲜有报道。跨膜蛋白14家族(Transmembrane 14 family, Tmemb14 family)是一个新发现的跨膜蛋白家族,目前只有拟南芥FAX1 (Fatty Acid Export 1)和斑马鱼TMEM14已被克隆鉴定,该家族其他成员的生物学功能还未见报道。AtFAX1参与植物质体长链脂肪酸的跨膜外运,其功能丧失显著降低植物生物量并影响花粉发育和育性。本研究通过生物信息手段对拟南芥和水稻中的跨膜蛋白14家族成员的进化关系、蛋白理化性质、结构域功能和编码基因的表达模式进行了分析,揭示了Tmemb14家族成员在单、双子叶植物进化中的功能分化,为进一步研究跨膜蛋白14家族成员的生理功能提供了理论依据。     

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.      

P-glycoprotein structure and evolutionary homologies

Analysis of multidrug resistant cell lines has led to the identification of the P-glycoprotein multigene family. Two of the three classes of mammalian P-glycoproteins have the ability to confer cellular resistance to a broad range of structurally and functionally diverse cytotoxic agents. P-glycoproteins are integral membrane glycoproteins comprised of two similar halves, each consisting of six membrane spanning domains followed by a cytoplasmic domain which includes a nucleotide binding fold. The P-glycoprotein is a member of a large superfamily of transport proteins which utilize ATP to translocate a wide range of substrates across biological membranes. This superfamily includes transport complexes comprised of multicomponent systems, half P-glycoproteins and P-glycoprotein-like homologs which appear to require approximately 12 α-helical transmembrane domains and two nucleotide binding folds for substrate transport. P-glycoprotein homologs have been isolated and characterized from a wide range of species. Amino acid sequences, the similarities between the halves and intron/exon boundaries have been compared to understand the evolutionary origins of the P-glycoprotein. This revised version was published online in August 2006 with corrections to the Cover Date.     

A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria.

Seventeen fully sequenced and two partially sequenced extracytoplasmic proteins of purple, gram-negative bacteria constitute a homologous family termed the putative membrane fusion protein (MFP) family. Each such protein apparently functions in conjunction with a cytoplasmic membrane transporter of the ATP-binding cassette family, major facilitator superfamily, or heavy metal resistance/nodulation/cell division family to facilitate transport of proteins, peptides, drugs, or carbohydrates across the two membranes of the gram-negative bacterial cell envelope. Evidence suggests that at least some of these transport systems also function in conjunction with a distinct outer membrane protein. We report here that the phylogenies of these proteins correlate with the types of transport systems with which they function as well as with the natures of the substrates transported. Characterization of the MFPs with respect to secondary structure, average hydropathy, and average similarity provides circumstantial evidence as to how they may allow localized fusion of the two gram-negative bacterial cell membranes. The membrane fusion protein of simian virus 5 is shown to exhibit significant sequence similarity to representative bacterial MFPs.     

Amino acid composition analysis of human secondary transport proteins and implications for reliable membrane topology prediction

Secondary transporters in humans are a large group of proteins that transport a wide range of ions, metals, organic and inorganic solutes involved in energy transduction, control of membrane potential and osmotic balance, metabolic processes and in the absorption or efflux of drugs and xenobiotics. They are also emerging as important targets for development of new drugs and as target sites for drug delivery to specific organs or tissues. We have performed amino acid composition (AAC) and phylogenetic analyses and membrane topology predictions for 336 human secondary transport proteins and used the results to confirm protein classification and to look for trends and correlations with structural domains and specific substrates and/or function. Some proteins showed statistically high contents of individual amino acids or of groups of amino acids with similar physicochemical properties. One recurring trend was a correlation between high contents of charged and/or polar residues with misleading results in predictions of membrane topology, which was especially prevalent in Mitochondrial Carrier family proteins. We demonstrate how charged or polar residues located in the middle of transmembrane helices can interfere with their identification by membrane topology tools resulting in missed helices in the prediction. Comparison of AAC in the human proteins with that in 235 secondary transport proteins from Escherichia coli revealed similar overall trends along with differences in average contents for some individual amino acids and groups of similar amino acids that are presumed to result from a greater number of functions and complexity in the higher organism.     

MIPDB: a relational database dedicated to MIP family proteins

BACKGROUND INFORMATION: The MIPs (major intrinsic proteins) constitute a large family of membrane proteins that facilitate the passive transport of water and small neutral solutes across cell membranes. Since water is the most abundant molecule in all living organisms, the discovery of selective water-transporting channels called AQPs (aquaporins) has led to new knowledge on both the physiological and molecular mechanisms of membrane permeability. The MIPs are identified in Archaea, Bacteria and Eukaryota, and the rapid accumulation of new sequences in the database provides an opportunity for large-scale analysis, to identify functional and/or structural signatures or to infer evolutionary relationships. To help perform such an analysis, we have developed MIPDB (database for MIP proteins), a relational database dedicated to members of the MIP family. RESULTS: MIPDB is a motif-oriented database that integrates data on 785 MIP proteins from more than 200 organisms and contains 230 distinct sequence motifs. MIPDB proposes the classification of MIP proteins into three functional subgroups: AQPs, glycerol-uptake facilitators and aquaglyceroporins. Plant MIPs are classified into three specific subgroups according to their subcellular distribution in the plasma membrane, tonoplast or the symbiosome membrane. Some motifs of the database are highly selective and can be used to predict the transport function or subcellular localization of unknown MIP proteins. CONCLUSIONS: MIPDB offers a user-friendly and intuitive interface for a rapid and easy access to MIP resources and to sequence analysis tools. MIPDB is a web application, publicly accessible at http://idefix.univ-rennes1.fr:8080/Prot/index.html.     

Sequence analyses of cyanobacterial bicarbonate transporters and their homologues

The primary HCO3- uptake system in the cyanobacterium Synecocystis is the Na+-dependent transporter SbtA. SbtA and its homologues were identified and shown to display a common topology of ten transmembrane segments (TMSs). These proved to have arisen by an intragenic duplication event from an ancestral gene encoding a five TMS protein product. A region of SbtA shows sufficient similarity to 10 TMS ABC-type integral membrane transport proteins to suggest a common origin. Phylogenetic analyses of the SbtA family revealed two clusters of cyanobacterial homologues with all non-cyanobacterial family members outside of these two clusters. The tree topology suggests that SbtA family members display multiple transport functions.     

Emerging new paradigms for ABCG transporters

Every cell is separated from its external environment by a lipid membrane. Survival depends on the regulated and selective transport of nutrients, waste products and regulatory molecules across these membranes, a process that is often mediated by integral membrane proteins. The largest and most diverse of these membrane transport systems is the ATP binding cassette (ABC) family of membrane transport proteins. The ABC family is a large evolutionary conserved family of transmembrane proteins (> 250 members) present in all phyla, from bacteria to Homo sapiens, which require energy in the form of ATP hydrolysis to transport substrates against concentration gradients. In prokaryotes the majority of ABC transporters are involved in the transport of nutrients and other macromolecules into the cell. In eukaryotes, with the exception of the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7), ABC transporters mobilize substrates from the cytoplasm out of the cell or into specific intracellular organelles. This review focuses on the members of the ABCG subfamily of transporters, which are conserved through evolution in multiple taxa. As discussed below, these proteins participate in multiple cellular homeostatic processes, and functional mutations in some of them have clinical relevance in humans.     

P-glycoprotein structure and evolutionary homologies

Analysis of multidrug resistant cell lines has led to the identification of the P-glycoprotein multigene family. Two of the three classes of mammalian P-glycoproteins have the ability to confer cellular resistance to a broad range of structurally and functionally diverse cytotoxic agents. P-glycoproteins are integral membrane glycoproteins comprised of two similar halves, each consisting of six membrane spanning domains followed by a cytoplasmic domain which includes a nucleotide binding fold. The P-glycoprotein is a member of a large superfamily of transport proteins which utilize ATP to translocate a wide range of substrates across biological membranes. This superfamily includes transport complexes comprised of multicomponent systems, half P-glycoproteins and P-glycoprotein-like homologs which appear to require 12 -helical transmembrane domains and two nucleotide binding folds for substrate transport. P-glycoprotein homologs have been isolated and characterized from a wide range of species. Amino acid sequences, the similarities between the halves and intron/exon boundaries have been compared to understand the evolutionary origins of the P-glycoprotein.     

Intra-helical salt-bridge and helix destabilizing residues within the same helical turn: Role of functionally important loop E half-helix in channel regulation of major intrinsic proteins

The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10–15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport.     

Structure and transmembrane topology of Slc11a1 TMD1-5 in lipid membranes

The importance of solute carrier family 11 (Slc11) in divalent metal-ion transport has been well established. The core domains TMD1-5 and TMD6-10 of the proteins were modeled as a symmetric but inversely orientated arrangement with respect to membrane normal. In this article, the structures and transmembrane topologies of TMD1-5 of Slc11a1 incorporated with phospholipids 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (POPG), and POPC/POPG (3:1) were explored using circular dichroism, fluorescence, and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopies. The segments TMD2-5 were inserted in lipid membranes mainly as an α-helix with orientations of helices along membrane normal. The tilt angles of the helices were in an order of TMD3 > TMD4 > TMD2 > TMD5 in these membranes. In contrast, TMD1 was partly inserted in membranes, leaving partial segment at membrane surface. The amount of the lipid component with negatively charged headgroups had an effect on both the helicity and orientation of the transmembrane domains (TMDs). Nevertheless, the helices maintained similar topologies in various membranes.     

Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.     

High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells

Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin; the lectins were visualized with protein-gold complexes. Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane. A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer. Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton.     

SWEET蛋白家族研究进展

SWEET是新发现的一类具有7次跨膜?-螺旋的糖运输蛋白,它们由2个重复的具有3次跨膜?-螺旋的MtN3 motif和一个起连接作用的跨膜?-螺旋组成.SWEET广泛存在于真核单细胞生物、高等植物以及动物中.它们在生殖发育、植物与微生物的相互作用、植物的逆境反应及衰老等许多方面起重要作用.最近的研究显示,原核生物中存在与真核生物SWEET类似的、只含有一个3次跨膜?-螺旋的蛋白,这些蛋白属于MtN3或PQ-Loop家族.从慢生根瘤菌中克隆的SWEET同源蛋白BjSemiSWEET1和已经鉴定的部分真核生物SWEET蛋白一样具有运输蔗糖的能力,这个结果与其他相关研究一起暗示真核生物7次跨膜?-螺旋的糖或氨基酸运输蛋白可能由原核生物中3次跨膜?-螺旋的小分子蛋白通过复制或横向基因转移融合进化而来,并且它们在行使功能时可能形成和其他许多膜转运蛋白相似的、具有12次跨膜结构的功能单位.对SWEET的研究将为揭示多种生命现象提供重要线索.     

Buried water molecules in helical transmembrane proteins

Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules.