A conserved WXXE motif is an apical delivery determinant of ABC transporter C subfamily isoforms.

ATP-binding cassette transporter isoform C7 (ABCC7), also designated as cystic fibrosis transmembrane conductance regulator (CFTR), is exclusively targeted to the apical plasma membrane of polarized epithelial cells. Although the apical localization of ABCC7 in epithelia is crucial for the Cl- excretion into lumens, the mechanism regulating its apical localization is poorly understood. In the present study, an apical localization determinant was identified in the N-terminal 80-amino acid long cytoplasmic region of ABCC7 (NT80). In HepG2 cells, overexpression of NT80 significantly disturbed the apical expression of ABCC7 in a competitive manner, suggesting the presence of a sorting determinant in this region. Deletion analysis identified a potential sorting information within a 20-amino acid long peptide (aa 41-60) of NT80. Alanine scanning mutagenesis of this region in full-length ABCC7 further narrowed down the apical localization determinant to four amino acids, W57DRE60. This WDRE sequence was conserved among vertebrate ABCC7 orthologs. Site-directed mutagenesis showed that W57 and E60 were critical for the apical expression of ABCC7, confirming a novel apical sorting determinant of ABCC7. Furthermore, a WXXE motif (tryptophan and glutamic acid residues with two-amino acid spacing) was found to be conserved among the N-terminal regions of apically localized ABCC members with 12-TM configuration. The significance of the WXXE motif was demonstrated for proper trafficking of ABCC4 to the apical plasma membrane.Key words: apical plasma membrane, sorting, ATP-binding cassette transporter, CFTR, MRP4.

Interaction mapping of the Sec61 translocon identifies two Sec61α regions interacting with hydrophobic segments in translocating chains.

Many proteins in organelles of the secretory pathway, as well as secretory proteins, are translocated across and inserted into the endoplasmic reticulum membrane by the Sec61 translocon, a protein-conducting channel. The channel consists of 10 transmembrane (TM) segments of the Sec61α subunit and possesses an opening between TM2b and TM7, termed the lateral gate. Structural and biochemical analyses of complexes of Sec61 and its ortholog SecY have revealed that the lateral gate is the exit for signal sequences and TM segments of translocating polypeptides to the lipid bilayer and also involved in the recognition of such hydrophobic sequences. Moreover, even marginally hydrophobic (mH) segments insufficient for membrane integration can be transiently stalled in surrounding Sec61α regions and cross-linked to them, but how the Sec61 translocon accommodates these mH segments remains unclear. Here, we used Cys-scanned variants of human Sec61α expressed in cultured 293-H cells to examine which channel regions associate with mH segments. A TM segment in a ribosome-associated polypeptide was mainly cross-linked to positions at the lateral gate, whereas an mH segment in a nascent chain was cross-linked to the Sec61α pore-interior positions at TM5 and TM10, as well as the lateral gate. Of note, cross-linking at position 180 in TM5 of Sec61α was reduced by an I179A substitution. We therefore conclude that at least two Sec61α regions, the lateral gate and the pore-interior site around TM5, interact with mH segments and are involved in accommodating them.

Simultaneous Measurement of Ear Canal Movement, Electromyography of the Masseter Muscle and Occlusal Force for Earphone-Type Occlusal Force Estimation Device Development.

We intend to develop earphone-type wearable devices to measure occlusal force by measuring ear canal movement using an ear sensor that we developed. The proposed device can measure occlusal force during eating. In this work, we simultaneously measured the ear canal movement (ear sensor value), the surface electromyography (EMG) of the masseter muscle and the occlusal force six times from five subjects as a basic study toward occlusal force meter development. Using the results, we investigated the correlation coefficient between the ear sensor value and the occlusal force, and the partial correlation coefficient between ear sensor values. Additionally, we investigated the average of the partial correlation coefficient and the absolute value of the average for each subject. The absolute value results indicated strong correlation, with correlation coefficients exceeding 0.9514 for all subjects. The subjects showed a lowest partial correlation coefficient of 0.6161 and a highest value of 0.8286. This was also indicative of correlation. We then estimated the occlusal force via a single regression analysis for each subject. Evaluation of the proposed method via the cross-validation method indicated that the root-mean-square error when comparing actual values with estimates for the five subjects ranged from 0.0338 to 0.0969.

The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel.

Voltage-dependent K+ (KV) channels control K+ permeability in response to shifts in the membrane potential. Voltage sensing in KV channels is mediated by the positively charged transmembrane domain S4. The best-characterized KV channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K+ channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp72 in S2 and Glu93 in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K+ channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP.

In vivo support for the new concept of pulmonary blood flow-mediated CO2 gas excretion in the lungs.

To further examine the validity of the proposed concept of pulmonary blood flow-dependent CO2 gas excretion in the lungs, we investigated the effects of intramediastinal balloon catheterization-, pulmonary artery catheterization-, or isoprenaline (ISP)-induced changes in pulmonary blood flow on the end-expiratory CO2 gas pressure (PeCO2 ), the maximal velocity of the pulmonary artery (Max Vp), systemic arterial pressure, and heart rate of anesthetized rabbits. We also evaluated the changes in the PeCO2 in clinical models of anemia or pulmonary embolism. An almost linear relationship was detected between the PeCO2 and Max Vp. In an experiment in which small pulmonary arteries were subjected to stenosis, the PeCO2 fell rapidly, and the speed of the reduction was dependent on the degree of stenosis. ISP produced significant increases in the PeCO2 of the anesthetized rabbits. Conversely, treatment with piceatannol or acetazolamide induced significant reductions in the PeCO2 . Treatment with a cell surface F1/FO ATP synthase antibody caused significant reductions in the PeCO2 itself and the ISP-induced increase in the PeCO2 . Neither the PeCO2 nor SAP was significantly influenced by marked anemia [%hematocrit (Ht), 70 ∼ 47%]. On the other hand, in the presence of less severe anemia (%Ht: 100 ∼ 70%) both the PeCO2 and SAP fell significantly when the rabbits' blood viscosity was decreased. The rabbits in which pulmonary embolisms were induced demonstrated significantly reduced PeCO2 values, which was compatible with the lowering of their Max Vp. In conclusion, we reaffirm the validity of the proposed concept of CO2 gas exchange in the lungs.

A few positively charged residues slow movement of a polypeptide chain across the endoplasmic reticulum membrane.

Many polypeptide chains are translocated across and integrated into the endoplasmic reticulum membrane through protein-conducting channels. During the process, amino acid sequences of translocating polypeptide chains are scanned by the channels and classified to be retained in the membrane or translocated into the lumen. We established an experimental system with which the kinetic effect of each amino acid residue on the polypeptide chain movement can be analyzed with a time resolution of tens of seconds. Positive charges greatly slow movement; only two lysine residues caused a remarkable slow down, and their effects were additive. The lysine residue was more effective than arginine. In contrast, clusters comprising three residues of each of the other 18 amino acids had little effect on chain movement. We also demonstrated that a four lysine cluster can exert the effect after being fully exposed from the ribosome. We concluded that as few as two to three residues of positively charged amino acids can slow the movement of the nascent polypeptide chain across the endoplasmic reticulum membrane. This effect provides a fundamental basis of the topogenic function of positively charged amino acids.

A cis-acting five-amino-acid motif controls targeting of ABCC2 to the apical plasma membrane domain.

ATP-binding cassette transporter isoform C2 (ABCC2) is exclusively targeted to the apical plasma membrane of polarized cells. Although apical localization of ABCC2 in hepatocytes is crucial for the biliary excretion of a variety of metabolites, the mechanism regulating its apical targeting is poorly understood. In the present study, an apical targeting signal was identified in the first cytoplasmic loop domain (CLD1) of ABCC2 in HepG2 cells. Overexpression of CLD1 significantly disturbed the apical targeting of FLAG-ABCC2 in a competitive manner, suggesting the presence of a saturable sorting machinery in HepG2 cells. Next, deletion analysis identified a potential targeting sequence within a 20-amino-acid long peptide (aa 272-291) of CLD1. Alanine scanning mutagenesis of this region in full-length ABCC2 further narrowed down the apical targeting determinant to five amino acids, S(283)QDAL(287). Of these, S(283) and L(287) were found to be conserved among vertebrate ABCC2 orthologs. Site-directed mutagenesis showed that both S(283) and L(287) were crucial for the targeting specificity of ABCC2. Introducing this apical targeting sequence into the corresponding region of ABCC1, an exclusively basolateral protein, caused the hybrid ABCC1 to partially localize in the apical membrane. Thus, the CLD1 of ABCC2 contains a novel apical sorting determinant, and a saturable sorting machinery is present in polarized HepG2 cells.

Positive charges on the translocating polypeptide chain arrest movement through the translocon.

Polypeptide chains synthesized by membrane-bound ribosomes are translocated through, and integrated into, the endoplasmic reticulum (ER) membrane by means of the protein translocation channel, the translocon. Positive charges on the nascent chain determine the orientation of the hydrophobic segment as it is inserted into the translocon and enhance the stop-translocation of translocating hydrophobic segments. Here we show that positive charges temporarily arrested ongoing polypeptide chain movement through the ER translocon by electrostatic interaction, even in the absence of a hydrophobic segment. The C-terminus of the polypeptide chain was elongated during the arrest, and then the full-length polypeptide chain moved through the translocon. The translocation-arrested polypeptide was not anchored to the membrane and the charges were on the cytoplasmic side of the membrane. The arrest effect was prevented by negatively charged residues inserted into the positive-charge cluster, and it was also suppressed by high salt conditions. We propose that positive charges are independent translocation regulators that are more active than previously believed.

Phosphatidylserine-binding protein lactadherin inhibits protein translocation across the ER membrane.

Secretory and membrane proteins are translocated across and inserted into the endoplasmic reticulum membrane via translocon channels. To investigate the effect of the negatively-charged phospholipid phosphatidylserine on the translocation of nascent polypeptide chains through the translocon, we used the phosphatidylserine-binding protein lactadherin C2-domain. Lactadherin inhibited targeting of nascent chain to the translocon by signal sequence and the initiation of translocation. Moreover, lactadherin inhibited the movement of the translocating polypeptide chain regardless of the presence or absence of positively-charged residues. Phosphatidylserine might be critically involved in translocon function, but it is not a major determinant for translocation arrest of positively-charged residues.

Involvement of a di-leucine motif in targeting of ABCC1 to the basolateral plasma membrane of polarized epithelial cells.

Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 to ABCC2 revealed that ABCC1 possesses a characteristic sequence, E(295)EVEALI(301), which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L(300) and I(301) residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E(295), E(296), and E(298) residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1.

Tail-extension following the termination codon is critical for release of the nascent chain from membrane-bound ribosomes in a reticulocyte lysate cell-free system.

Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors.

Pleiotropic effects of membrane cholesterol upon translocation of protein across the endoplasmic reticulum membrane.

Various proteins are translocated through and inserted into the endoplasmic reticulum membrane via translocon channels. The hydrophobic segments of signal sequences initiate translocation, and those on translocating polypeptides interrupt translocation to be inserted into the membrane. Positive charges suppress translocation to regulate the orientation of the signal sequences. Here, we investigated the effect of membrane cholesterol on the translocational behavior of nascent chains in a cell-free system. We found that the three distinct translocation processes were sensitive to membrane cholesterol. Cholesterol inhibited the initiation of translocation by the signal sequence, and the extent of inhibition depended on the signal sequence. Even when initiation was not inhibited, cholesterol impeded the movement of the positively charged residues of the translocating polypeptide chain. In surprising contrast, cholesterol enhanced the translocation of hydrophobic sequences through the translocon. On the basis of these findings, we propose that membrane cholesterol greatly affects partitioning of hydrophobic segments into the membrane and impedes the movement of positive charges.

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins.

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH(2)-terminal deletion constructs of P70R. Among the NH(2)-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH(2)-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH(2)-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH(2)-terminal hydrophobic TMS1. Furthermore, attachment of the NH(2)-terminal hydrophilic domain to the NH(2)-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH(2)-terminal region determines the subcellular localization of ABC subfamily D proteins.

Eyes Closing and Drowsiness in Human Subjects Decrease Baseline Galvanic Skin Response and Active Palmar Sweating: Relationship Between Galvanic Skin and Palmar Perspiration Responses.

We previously constructed a perspiration ratemeter for the measurement of palmar sweating in human subjects. Although galvanic skin response (GSR) has been used to evaluate emotional responses in human subjects, little is known about the relationships between the phasic and baseline components in GSR and active palmar sweating. From the aforementioned, we aimed to investigate the relationships in human subjects with handgrip exercise and eyes closing or opening. Fifteen healthy volunteers (mean age: 26.9 ± 8.7 years) participated in the present experiments. We investigated the effects of maximal handgrip exercise, eyes closing or opening, and self-awareness of drowsy on the GSR, active palmar sweating, R-R interval in electrocardiograph (ECG), and percentage of α wave in EEG. The faster phasic component in GSR completely agreed with the starting point of active palmar sweating. Handgrip exercise induced significantly faster spike in GSR, active palmar sweating, and decrease in R-R interval in ECG. Eyes closing produced significant decreases in baseline GSR and active palmar sweating in all human subjects. The percentage of α wave in electroencephalograph (EEG) also increased. In contrast, eyes opening increased significantly the baseline GSR and active palmar sweating. In the equivalent electrical model of human skin, the eyes closing-mediated time-dependent decrease in the baseline GSR completely agreed with the hypothesis that the palmar skin voltage only in the model decreased time dependently to 0.4 of the control during 6 min. The self-awareness of drowsy in mid-night working with computer produced similar decreases in baseline GSR and active palmar sweating to the responses with eyes closing in all human subjects. In conclusion, the faster spike in GSR completely agreed with the starting point of active palmar sweating. Eyes closing and opening or self-awareness of drowsy significantly produced changes in baseline GSR and active palmar sweating, which may become useful tools for evaluating clearness or drowsiness in human subjects.

Human ABC transporter isoform B6 (ABCB6) localizes primarily in the Golgi apparatus.

Human ATP-binding cassette transporter isoform B6 (ABCB6) has been proposed to be situated in both the inner and outer membranes of mitochondria. These inconsistent observations of submitochondrial localization have led to conflicting interpretation in view of directions of transport facilitated by ABCB6. We show here that ABCB6 has an N-terminal hydrophobic region of 220 residues that functions as a primary determinant of co-translational targeting to the endoplasmic reticulum (ER), but it does not have any known features of a mitochondrial targeting sequence. We defined the potential role of this hydrophobic extension of ABCB6 by glycosylation site mapping experiments, and demonstrated that the first hydrophobic segment acts as a type I signal-anchor sequence, which mediates N-terminal translocation through the ER membrane. Laser scanning microscopic observation revealed that ABCB6 did not co-localize with mitochondrial staining. Rather, it localized in the ER-derived and brefeldin A-sensitive perinuclear compartments, mainly in the Golgi apparatus.

Ontogenic isoform switching of UDP-glucuronosyltransferase family 1 in rat liver.

The liver undergoes dramatic changes in function during development. The development of UDP-glucuronosyltransferase family 1 (UGT1) isoforms was studied in livers from rats at 16-20days of gestation, at days 1, 2, 3, 4, and 7 of infancy, at days 14 and 28 of childhood, and at day 56 of young adulthood. We found developmental stage-specific switching of regulation of the rat UGT1 gene complex. UGT1A6 was expressed as a predominant component of UGT1 in fetus liver, while other UGT1 isoforms were repressed. In contrast, expression of UGT1A1 surged immediately after birth. Expression of UGT1A5 was transiently elevated in childhood. We also found age-dependent alternative usage of dual UGT1A6 promoters in rat liver. Since UGT1A1 is the only bilirubin-glucuronidating isoform, the ontogeny of UGT1A1 in liver microsomes demonstrates that inadequate UGT1A1 proteins in the early neonatal period are linked to the common etiology of idiopathic hyperbilirubinemia in the newborn infant.

Switching the sorting mode of membrane proteins from cotranslational endoplasmic reticulum targeting to posttranslational mitochondrial import.

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.

Vestibular hair bundles control pH with (Na+, K+)/H+ exchangers NHE6 and NHE9.

In hair cells of the inner ear, robust Ca2+/H+ exchange mediated by plasma-membrane Ca2+-ATPase would rapidly acidify mechanically sensitive hair bundles without efficient removal of H+. We found that, whereas the basolateral membrane of vestibular hair cells from the frog saccule extrudes H+ via an Na+-dependent mechanism, bundles rapidly remove H+ in the absence of Na+ and HCO3(-), even when the soma is acidified. K+ was fully effective and sufficient for H+ removal; in contrast, Rb+ failed to support pH recovery. Na+/H+-exchanger isoform 1 (NHE1) was present on hair-cell soma membranes and was likely responsible for Na+-dependent H+ extrusion. NHE6 and NHE9 are organellar isoforms that can appear transiently on plasma membranes and have been proposed to mediate K+/H+ exchange. We identified NHE6 in a subset of hair bundles; NHE9 was present in all bundles. Heterologous expression of these isoforms in yeast strains lacking endogenous exchangers conferred pH-dependent tolerance to high levels of KCl and NaCl. NHE9 preferred cations in the order K+, Na+ >> Rb+, consistent with the relative efficacies of these ions in promoting pH recovery in hair bundles. Electroneutral K+/H+ exchange, which we propose is performed by NHE9 in hair bundles, exploits the high-K+ endolymph, responds only to pH imbalance across the bundle membrane, is unaffected by the +80 mV endocochlear potential, and uses mechanisms already present in the ear for K+ recycling. This mechanism allows the hair cell to remove H+ generated by Ca2+ pumping without ATP hydrolysis in the cell.

Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane.

We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the C-terminal five-residue segment (C-segment) contained the information required for mitochondrial-targeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.

Role of Pex19p in the targeting of PMP70 to peroxisome.

Pex19p is a protein required for the peroxisomal membrane synthesis. The 70-kDa peroxisomal membrane protein (PMP70) is synthesized on free cytosolic ribosomes and then inserted posttranslationally into peroxisomal membranes. Pex19p has been shown to play an important role in this process. Using an in vitro translation system, we investigated the role of Pex19p as a chaperone and identified the regions of PMP70 required for the interaction with Pex19p. When PMP70 was translated in the presence of purified Pex19p, a large part of PMP70 existed as soluble form and was co-immunoprecipitated with Pex19p. However, in the absence of Pex19p, PMP70 formed aggregates during translation. To identify the regions that interact with Pex19p, various truncated PMP70 were translated in the presence of Pex19p and subjected to co-immunoprecipitation. The interaction was markedly reduced by the deletion of the NH(2)-terminal 61 amino acids or the region around TMD6. Further, we expressed these deletion constructs of PMP70 in fusion with the green fluorescent protein in CHO cells. Fusion proteins lacking these Pex19p binding sites did not display any peroxisomal localization. These results suggest that Pex19p binds to PMP70 co-translationally and keeps PMP70 as a proper conformation for the localization to peroxisome.