Equilibrative nucleoside transporters: mapping regions of interaction for the substrate analogue nitrobenzylthioinosine (NBMPR) using rat chimeric proteins.

The rat equilibrative nucleoside transporters rENT1 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine (NBMPR). Structurally, the proteins have a large glycosylated extracellular loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, we have generated chimeras between NBMPR-sensitive rENT1 and NBMPR-insensitive rENT2, using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) to identify structural domains of rENT1 responsible for transport inhibition by NBMPR. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 NBMPR-insensitive. Domain swaps within the amino-terminal halves of rENT1 and rENT2 identified two contiguous regions, TMs 3-4 (rENT1 residues 100-171) and TMs 5-6 (rENT1 residues 172-231), as the major sites of NBMPR interaction. Since NBMPR is a nucleoside analogue and functions as a competitive inhibitor of zero-trans nucleoside influx, TMs 3-6 are likely to form parts of the substrate translocation channel.     

More Than 1,001 Problems with Protein Domain Databases: Transmembrane Regions,Signal Peptides and the Issue of Sequence Homology

Large-scale genome sequencing gained general importance for life science because functional annotation of otherwise experimentally uncharacterized sequences is made possible by the theory of biomolecular sequence homology. Historically, the paradigm of similarity of protein sequences implying common structure, function and ancestry was generalized based on studies of globular domains. Having the same fold imposes strict conditions over the packing in the hydrophobic core requiring similarity of hydrophobic patterns. The implications of sequence similarity among non-globular protein segments have not been studied to the same extent; nevertheless, homology considerations are silently extended for them. This appears especially detrimental in the case of transmembrane helices (TMs) and signal peptides (SPs) where sequence similarity is necessarily a consequence of physical requirements rather than common ancestry. Thus, matching of SPs/TMs creates the illusion of matching hydrophobic cores. Therefore, inclusion of SPs/TMs into domain models can give rise to wrong annotations. More than 1001 domains among the 10,340 models of Pfam release 23 and 18 domains of SMART version 6 (out of 809) contain SP/TM regions. As expected, fragment-mode HMM searches generate promiscuous hits limited to solely the SP/TM part among clearly unrelated proteins. More worryingly, we show explicit examples that the scores of clearly false-positive hits, even in global-mode searches, can be elevated into the significance range just by matching the hydrophobic runs. In the PIR iProClass database v3.74 using conservative criteria, we find that at least between 2.1% and 13.6% of its annotated Pfam hits appear unjustified for a set of validated domain models. Thus, false-positive domain hits enforced by SP/TM regions can lead to dramatic annotation errors where the hit has nothing in common with the problematic domain model except the SP/TM region itself. We suggest a workflow of flagging problematic hits arising from SP/TM-containing models for critical reconsideration by annotation users.     

LMP1 transmembrane domain 1 and 2 (TM1-2) FWLY mediates intermolecular interactions with TM3-6 to activate NF-kappaB

The Epstein-Barr virus oncoprotein LMP1 has six transmembrane domains (TMs) that enable intermolecular aggregation and constitutive signaling through two C-terminal cytosolic domains. Expression of both TMs 1 and 2 without the C terminus (TM1-2DeltaC) and TMs 3 to 6 fused to the C terminus (TM3-6) results in partial association, which is substantially decreased by TM1 F38WLY41 mutation to A38ALA41. We now investigate whether TM1-2DeltaC can functionally interact with TM3-6. TM1-2DeltaC induced TM3-6 to mediate NF-kappaB activation at 59% of LMP1 levels, and the effect was dependent on TM1-2 F38WLY41. TM1-2DeltaC even induced TM3-4 C terminus-mediated NF-kappaB activation to 44% of LMP1 levels. Surprisingly, this effect was TM1 F38WLY41 independent, indicative of a role for TMs 5 and 6 in TM1 F38WLY41 effects. TM3 W98 was also important for TM1-2DeltaC induction of TM3-6-mediated NF-kappaB activation, for association, and for TM1 F38WLY41 dependence on C-terminal NF-kappaB activation. These data support models in which the TM1 F38WLY41 effects are at least partially dependent on TM3 W98 and a residue(s) in TMs 5 and 6.     

Mutational and sequence analysis of transmembrane segment 6 orientation in TetA proteins

The packing orientations of the 8 transmembrane (TM) segments that line the central, aqueous transport channel within tetracycline resistance proteins (TetA) have been established. However, the orientations of the remaining 4 segments, TMs 3, 6, 9, and 12, located at the periphery, and away from the transport channel, have not yet been determined. In this study, the packing orientation of TM6 within the class C TetA protein encoded by plasmid pBR322 was evaluated by substitution mutagenesis and analysis of sequence conservation and amphipathicity. The combined data support a model in which the conserved and polar face of the TM6 alpha-helix containing Asn170 and Asn173 orients towards channel-lining TM segments, and the relatively non-conserved and hydrophobic face of TM6 points towards membrane lipids.     

Surface expression of mutated subunits of the high affinity mast cell receptor for IgE

The mast cell receptor with high affinity for IgE consists of four transmembrane polypeptides which are held together by detergent-sensitive interactions: an IgE-binding alpha chain, a single beta chain, and a disulfide-linked dimer of gamma chains. Now that the cDNAs that code for each of the subunits have been isolated, it should be possible to probe by site-specific mutations, which portions of the receptor are critical for transmembrane signaling. One prerequisite for such studies is that the mutant receptors be expressible on the cell surface. We have explored this issue by transiently transfecting COS 7 cells with mutant subunits and assessing surface expression by IgE binding. Removal of any single cytoplasmic domain of the receptor's subunits had little influence on surface expression, and even receptors missing all five cytoplasmic domains were expressed, albeit less efficiently. Minor changes within the transmembrane domains (TMs) sometimes produced major effects and more drastic changes in the TMs ablated surface expression entirely. These data suggest that the TMs are critical loci for receptor display. Cys7 (residue 2 in the gamma TM) was shown to form the inter-gamma disulfide bond and to be nonessential for surface expression. By localizing this bond, residues in the TM of gamma that are buried in the interface between the gamma subunits could be predicted. Consistent with observations on other membrane proteins (Rees, D. C., DeAntonio, L., and Eisenberg, D. (1989) Science 245, 510-513), maximal interspecies conservation was observed for those residues in the gamma TM predicted to be buried. This was also true for those residues in the alpha and beta TMs predicted to be buried by analysis of the TM hydrophobic moments.     

Converting a marginally hydrophobic soluble protein into a membrane protein

δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.     

The complex process of GETting tail-anchored membrane proteins to the ER

Biosynthesis of membrane proteins requires that hydrophobic transmembrane (TM) regions be shielded from the cytoplasm while being directed to the correct membrane. Tail-anchored (TA) membrane proteins, characterized by a single C-terminal TM, pose an additional level of complexity because they must be post-translationally targeted. In eukaryotes, the GET pathway shuttles TA-proteins to the endoplasmic reticulum. The key proteins required in yeast (Sgt2 and Get1-5) have been under extensive structural and biochemical investigation during recent years. The central protein Get3 utilizes nucleotide linked conformational changes to facilitate substrate loading and targeting. Here we analyze this complex process from a structural perspective, as understood in yeast, and further postulate on similar pathways in other domains of life.     

Membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase Doa10 and comparison with its human ortholog TEB4 (MARCH-VI)

Quality control machinery in the endoplasmic reticulum (ER) helps ensure that only properly folded and assembled proteins accumulate in the ER or continue along the secretory pathway. Aberrant proteins are retrotranslocated to the cytosol and degraded by the proteasome, a process called ER-associated degradation. Doa10, a transmembrane protein of the ER/nuclear envelope, is one of the primary ubiquitin ligases (E3s) participating in ER-associated degradation in Saccharomyces cerevisiae. Here we report the membrane organization of the 1319-residue Doa10 polypeptide. The topology was determined by fusing a dual-topology reporter after 16 different Doa10 fragments. Our results indicate that Doa10 contains 14 transmembrane helices (TMs). Based on protease digestion of yeast microsomes, both the N-terminal RING-CH domain and the C terminus face the cytosol. Notably, the experimentally derived topology was not predicted correctly by any of the generally available TM prediction algorithms. Bioinformatic analysis and in silico mutagenesis guided the topological studies through problematic regions. The conserved TD domain in Doa10 includes three TMs. These TMs might function in cofactor binding or substrate recognition, or they might be part of a retrotranslocation channel. The Derlins were previously proposed to provide such channels, but we show that the two yeast Derlins are not required for degradation of Doa10 membrane substrates, as was found before for the Sec61 translocon. Finally, we provide evidence that the likely human Doa10 ortholog, TEB4 (MARCH-VI), adopts a topology similar to that of Doa10.     

Membrane topography and topogenesis of prenylated Rab acceptor (PRA1)

The mouse prenylated Rab acceptor (mPRA1) is associated with the Golgi membrane at steady state and interacts with Rab proteins. It contains two internal hydrophobic domains (34 residues each) that have enough residues to form four transmembrane (TM) segments. In this study, we have determined the membrane topography of mPRA1 in both intact cells and isolated microsomes. The putative TM segments of mPRA1 were used to substitute for a known TM segment of a model membrane protein to determine whether the mPRA1 segments integrate into the membrane. Furthermore, N-linked glycosylation scanning methods were used to distinguish luminal domains from cytoplasmic domains of mPRA1. The data demonstrate that mPRA1 is a polytopic membrane protein containing four TM segments. These TM segments act cooperatively during the translocation and integration at the endoplasmic reticulum membrane. All hydrophilic domains are in the cytoplasm, including the N-terminal domain, the linker domain between the two hydrophobic domains, and the C-terminal domain. As a result, the bulk of mPRA1 is located in the cytoplasm, supporting its postulated role in regulating Rab membrane targeting and intracellular trafficking.     

Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein

We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the alpha and beta subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61alpha and Sec61beta during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the "stage" of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.     

The two membrane segments of leader peptidase partition one by one into the lipid bilayer via a Sec/YidC interface

We have addressed the mechanism of insertion of both transmembrane segments (TMs) of leader peptidase, a double-spanning protein, into the Escherichia coli inner membrane. Using photo-crosslinking, the first TM (H1) was shown to insert at a Sec-translocon/YidC interface in a fixed orientation. H1 lost its contacts with the Sec-translocon and gained access to lipids near YidC soon after complete exposure outside the ribosome. Following lipid integration, it moved away from the Sec/YidC insertion site. The second TM (H2) inserted and interacted with SecY and YidC in a similar transient fashion. The data are consistent with a linear integration model in which the TMs of polytopic inner membrane proteins move one by one from a Sec/YidC insertion site into the lipid bilayer. We propose that YidC assists the lipid partitioning of single TMs.     

Evidence for similar function of transmembrane segments in receptor and membrane-anchored proteins

Transmembrane (TM) regions of receptor proteins should have unique structural and/or chemical characteristics if these regions contain residues functional in TM signal transduction. However, in a survey of the membrane-occurring residues in 37 integral membrane proteins, we found that amino acid compositions of TM regions of receptor proteins (n = 11) could not be distinguished statistically from corresponding regions of membrane-anchored proteins (e.g., recognition molecules) with a functional external domain attached to a single hydrophobic membrane-spanning anchor segment (n = 16). TM regions in both categories of proteins differed from the compositions of TM regions in membrane-transport proteins (n = 10). The analysis implies that TM regions in receptor proteins may function mainly to anchor (and position) receptors in their cellular membranes, and therefore residues in receptors that participate in signal transduction need not be restricted to these regions. In addition to mechanisms involving receptor aggregation, ligand-activated conformational perturbation of a receptor external aqueous domain, resulting in membrane penetration of hydrophobic segment(s) of this domain to produce intramembranous contact with its cytoplasmic domain, is hypothesized as a further possible mode of signal transduction.     

A knowledge-based scale for the analysis and prediction of buried and exposed faces of transmembrane domain proteins

MOTIVATION: The dearth of structural data on alpha-helical membrane proteins (MPs) has hampered thus far the development of reliable knowledge-based potentials that can be used for automatic prediction of transmembrane (TM) protein structure. While algorithms for identifying TM segments are available, modeling of the TM domains of alpha-helical MPs involves assembling the segments into a bundle. This requires the correct assignment of the buried and lipid-exposed faces of the TM domains. RESULTS: A recent increase in the number of crystal structures of alpha-helical MPs has enabled an analysis of the lipid-exposed surfaces and the interiors of such molecules on the basis of structure, rather than sequence alone. Together with a conservation criterion that is based on previous observations that conserved residues are mostly found in the interior of MPs, the bias of certain residue types to be preferably buried or exposed is proposed as a criterion for predicting the lipid-exposed and interior faces of TMs. Applications to known structures demonstrates 80% accuracy of this prediction algorithm. AVAILABILITY: The algorithm used for the predictions is implemented in the ProperTM Web server (http://icb.med.cornell.edu/services/propertm/start).     

Functional assembly of the Na+/H+ antiporter of Helicobacter pylori from partial fragments in vivo

Functional assembly of the Helicobacter pylori Na+/H+ antiporter (HPNhaA) from partial fragments was studied. Expression plasmids encoding a series of complementary N- and C-terminal fragment pairs containing the transmembrane domains (TMs) were constructed by inserting a stop or a start codon into each of the loop regions of NhaA. HPNhaA fragments alone or complementary fragment pairs were expressed in DeltanhaA Escherichia coli, and fragment integration into the membrane and antiporter activity were measured. TM1-10, TM1-11, TM2-12, TM6-12, and TM10-12 were found in the membrane fraction, while the other fragments were not. While no single fragment displayed antiporter activity, simultaneous expression of fragments in certain pairs, such as TM1-2 + TM3-12, TM1-8 + TM9-12, or TM1-11 + TM12, reconstituted antiporter activity. With the exception of TM12, all of the fragments in the pairs were detected in the membrane. No single fragments expressed alone for these pairs were found in the membrane, except for TM1-11, suggesting that the interaction between the fragments in these pairs stabilized the fragments and enabled reconstitution of HPNhaA. We also found that the simultaneous expression of three complementary fragments (TM1-2 + TM3-8 + TM9-12) reconstituted HPNhaA activity. Other pairs that were found in the membrane (TM1-5 + TM6-12, TM1-10 + TM11-12, and TM1 + TM2-12) did not reconstitute antiporter activity, suggesting that they may not have the proper conformation. These results revealed that the ability to reconstitute antiporter activity depends on the split position in the loop regions and the interaction between complementary fragment pairs. We propose that formation of the active HPNhaA molecule is initiated by the interaction of short-lived intermediates and maintained by the increased stability of the intermediates within the resulting complex.     

Unusual topological arrangement of structural motifs in the baboon reovirus fusion-associated small transmembrane protein

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N(exoplasmic)/C(cytoplasmic) topology. This topology results in the translocation of the smallest ectodomain ( approximately 20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated membrane fusion.     

Use of six chimeric proteins to investigate the role of intramolecular interactions in determining the kinetics of carnitine palmitoyltransferase I isoforms

The two isoforms of carnitine palmitoyltransferase I (CPT I; muscle (M)- and liver (L)-type) of the mitochondrial outer membrane have distinct kinetic characteristics with respect to their affinity for one of the substrates (l-carnitine) and the inhibitor malonyl-CoA. Moreover, they differ markedly in their hysteretic behavior with respect to malonyl-CoA and in their response to changes in the in vivo metabolic state. However, the two proteins are 62% identical and have the same overall structure. Using liver mitochondria, we have previously shown that the protein is polytopic within the outer membrane, comprising a 46-residue cytosolic N-terminal sequence, two transmembrane segments (TM1 and TM2) separated by a 27-residue loop, and a large catalytic domain (also cytosolic) (Fraser, F., Corstorphine, C. G., and Zammit, V. A. (1997) Biochem. J. 323, 711-718). We have now conducted a systematic study on six chimeric proteins constructed from combinations of three linear segments of rat L- and M-CPT I and on the two parental proteins to elucidate the effects of altered intramolecular interactions on the kinetics of CPT activity. The three segments were (i) the cytosolic N-terminal domain plus TM1, (ii) the loop plus TM2, and (iii) the cytosolic catalytic C-terminal domain. The kinetic properties of the chimeric proteins expressed in Pichia pastoris were studied. We found that alterations in the combinations of the N-terminal plus TM1 and C-terminal domains as well as in the N terminus plus TM1/TM2 pairings resulted in changes in the K(m) values for carnitine and palmitoyl-CoA and the sensitivity to malonyl-CoA of the L-type catalytic domain. The changes in affinity for malonyl-CoA and palmitoyl-CoA occurred independently of changes in the affinity for carnitine. The kinetic characteristics of the M-type catalytic domain and, in particular, its malonyl-CoA sensitivity were much less susceptible to influence by exchange of the other two segments of the protein. The marked difference in the response of the two catalytic domains to changes in the N-terminal domain and TM combinations explains the previously observed differences in the response of L- and M-CPT I to altered physiological state in intact mitochondria and to modulation of altered lipid molecular order of the mitochondrial outer membrane in vivo and in vitro.     

Isolation and characterization of cDNA clones encoding a low molecular weight nonmuscle tropomyosin isoform

cDNA clones encoding rat fibroblast tropomyosin 4 (TM-4) were isolated and characterized. DNA sequence analysis was carried out to determine the sequence of the protein. The derived amino acid sequence revealed that rat fibroblast TM-4 was found to contain 248 amino acids. The amino acid sequence of rat fibroblast TM-4 was compared with two other low molecular weight TM isoforms, equine platelet beta-TM and a human fibroblast TM. Rat TM-4 exhibited 98% sequence identity with the equine platelet TM but only 75% identity with the human fibroblast TM isoform. The high degree of conservation between the rat and equine proteins indicates that they belong to the same isotype of TM. Comparison of the amino acid sequences of the three low molecular TM isoforms along the length of the proteins reveals regions that are strongly conserved and regions that have considerably diverged. In the regions from amino acid residues 1 to 148 and 176 to 221, amino acid substitutes are moderate. The most variant regions in the sequence are in the middle part of the proteins from amino acids 149 to 175 and at the carboxyl-terminal region of the proteins from amino acids 222 to 248. The differences in the sequence of the rat and platelet TMs compared to the human TM may define distinct functional domains among the low molecular weight TMs. In addition, expression of tropomyosin was studied in a variety of tissues and transformed cells. We also demonstrate that at least three separate genes encode tropomyosins expressed in rat fibroblasts.     

Modeling and docking of the three-dimensional structure of the human melanocortin 4 receptor

A three-dimensional structure of the human melanocortin 4 receptor (hMC4R) is constructed in this study using a computer-aided molecular modeling approach. Human melanocortin 4 receptor is a G Protein-Coupled Receptor (GPCR). We structurally aligned transmembrane helices with bovine rhodopsin transmembrane domains, simulated both intracellular and extracellular loop domains on homologous loop regions in other proteins of known 3D structure and modeled the C terminus on the corresponding part of bovine rhodopsin. Then tandem minimization and dynamics calculations were run to refine the crude structure. The simulative model was tested by docking with a triplet peptide (RFF) ligand. It was found that the ligand is located among transmembrane regions TM3, TM4, TM5, and TM6 of hMC4R. In consistence with mutational and biochemical data, binding site is mainly formed as a hydrophobic and negatively charged pocket. The model constructed here might provide a structural framework for making rational predictions in relevant fields.     

Amino acid residues in transmembrane domain 10 of organic anion transporting polypeptide 1B3 are critical for cholecystokinin octapeptide transport

Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are multispecific transporters that mediate uptake of amphipathic organic compounds into hepatocytes. The two OATPs contain 12 transmembrane domains (TMs) and share 80% amino acid sequence identity. Besides common substrates with OATP1B1, OATP1B3 specifically transports cholecystokinin octapeptide (CCK-8). To determine which structural domains and/or residues are important for the substrate selectivity of OATP1B3, we constructed a series of chimeric proteins between OATP1B3 and 1B1, expressed them in HEK293 cells, and determined rates of uptake of CCK-8 along with surface expression of the proteins. Replacing TM10 in OATP1B3 with TM10 of OATP1B1 resulted in a dramatically reduced degree of CCK-8 transport, indicating that TM10 is crucial for recognition and/or translocation of CCK-8. Using site-directed mutagenesis, we identified three key residues within TM10, namely, Y537, S545, and T550. When we replaced these residues with the corresponding amino acid residues found in OATP1B1, the level of CCK-8 transport was similarly low as for the replacement of the whole TM10. Kinetic experiments showed that the K m values for CCK-8 transport in the TM10 replacement and triple mutant were only 1.3 and 1.1 microM, respectively, as compared to 16.3 microM for wild-type OATP1B3. Similarly, the V max values dropped from 495.5 pmol (normalized mg) (-1) min (-1) for wild-type OATP1B3 to 13.3 and 19.0 pmol (normalized mg) (-1) min (-1) for the TM10 replacement and triple mutant, respectively. Molecular modeling indicated that two of the three identified residues might form hydrogen bonds with CCK-8. In conclusion, we have identified three amino acid residues (Y537, S545, and T550) in TM10 of OATP1B3 that are important for CCK-8 transport.     

Properties of the intracellular transient receptor potential (TRP) channel in yeast, Yvc1

Transient receptor potential (TRP) channels are found among mammals, flies, worms, ciliates, Chlamydomonas, and yeast but are absent in plants. These channels are believed to be tetramers of proteins containing six transmembrane domains (TMs). Their primary structures are diverse with sequence similarities only in some short amino acid sequence motifs mainly within sequences covering TM5, TM6, and adjacent domains. In the yeast genome, there is one gene encoding a TRP-like sequence. This protein forms an ion channel in the vacuolar membrane and is therefore called Yvc1 for yeast vacuolar conductance 1. In the following we summarize its prominent features.