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.

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.

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.

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.

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.

Stability and flexibility of marginally hydrophobic-segment stalling at the endoplasmic reticulum translocon.

Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an Ncytosol/Clumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase.

The N-terminal motif of PMP70 suppresses cotranslational targeting to the endoplasmic reticulum.

Many membrane proteins possessing hydrophobic transmembrane (TM) segments are cotranslationally integrated into the endoplasmic reticulum (ER) membrane. Various peroxisomal and mitochondrial membrane proteins escape the ER-targeting mechanism and are targeted to their destinations. Here, we discovered a short segment in the 70-kDa peroxisomal membrane protein (PMP70) that suppresses ER targeting. The first TM segment has an intrinsic signal function that targets the nascent chain to the ER. The ER targeting was suppressed by a short N-terminal sequence of nine residues that is 80 residues upstream of the TM segment. Among the nine residues, Ser(5) is indispensable. The short segment also suppressed the signal peptide function of an authentic secretory protein. This function of the short segment was suppressed by the recombinant motif-GST fusion protein. The 50-kDa and 20-kDa proteins were crosslinked with the motif. The PMP70 molecule with the Ser5Ala point mutation predominantly localized to the ER. We propose the concept of an ER-targeting suppressor that suppresses the ER-targeting mechanism via a binding factor.

Capsid protease domain as a tool for assessing protein-domain folding during organelle import of nascent polypeptides in living cells.

Various proteins synthesized by ribosomes are imported into specific organelles. To elucidate the behavior of protein domains during import, we developed a folding probe, in which the capsid protease (CP) domain of the Semliki Forest virus was connected to enhanced green fluorescent protein (EGFP). The probe was fused to appropriate N-terminal organelle-targeting signal sequences and expressed in cultured cells. When the entire CP-domain was present in the cytosol, it became folded and cleaved off the following EGFP-domain. Once cleaved, EGFP stability was not affected by upstream sequences. Based on EGFP localization, we estimated the extent of CP-domain folding in the cytosolic space. When fused to mitochondrial hydrophobic multispanning membrane protein ABCB10, more than half of the EGFP remained in the cytoplasm, whereas most of the CP-portion was in the mitochondrial fraction. When fused to the endoplasmic reticulum (ER) signal, the cleaved EGFP was observed only in the ER fraction, confirming that the CP-domain cannot fold on the cytoplasmic side during cotranslational ER translocation. Thus, import of the ABCB10 molecule was not as tightly coupled with chain elongation as ER translocation. Use of this probe to quantitatively examine stop-translocation at the ER translocon in living cells revealed that positively charged residues on the translocating nascent chain stall at the ER translocon.

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.

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.

Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences.

Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10(-8) M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I-like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.

Stop-and-move of a marginally hydrophobic segment translocating across the endoplasmic reticulum membrane.

Many membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane via the protein-conducting channel, the so-called translocon. The hydrophobic transmembrane segment of the translocating nascent polypeptide chain stops at the translocon and then moves laterally into the membrane. Partitioning of the hydrophobic segment into the membrane is the primary determinant for membrane insertion. Here, we examined the behavior of a marginally hydrophobic segment at the translocon and found that its stop-translocation was greatly affected by the C-terminally attached ribosomes. The marginally hydrophobic segment first stops at the membrane and then moves into the lumen as long as the nascent chain is attached to translating ribosomes. When it is released from the ribosome by the termination codon, the marginally hydrophobic segment does not move. Puromycin or RNase treatment also suppressed movement. The movement was reversibly inhibited by high-salt conditions and irreversibly inhibited by ethylenediaminetetraacetic acid. There is an unstable state prior to the stable membrane insertion of the transmembrane segment. This characteristic state is maintained by the synthesizing ribosome.

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.

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.

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.

A sugar chain at a specific position in the nascent polypeptide chain induces forward movement during translocation through the translocon.

Nascent polypeptide chains synthesized by membrane bound ribosomes are cotranslationally translocated through and integrated into the endoplasmic reticulum translocon. Hydrophobic segments and positive charges on the chain are critical to halt the ongoing translocation. A marginally hydrophobic segment, which cannot be inserted into the membrane by itself, can be a transmembrane segment depending on its downstream positive charges. In certain conditions, positive charges even 60 residues downstream cause the marginally hydrophobic segment to span the membrane by inducing the segment to slide back from the lumen. Here we systematically examined the effect of a core sugar chain on the fate of a marginally hydrophobic segment using a cell-free translation and translocation system. A sugar chain added within 12 residues upstream of the marginally hydrophobic segment prevents the sliding back and promotes forward movement of the polypeptide chain. The sugar chain apparently functions as a ratchet to keep the polypeptide chain in the lumen. We propose that the sugar chain is a third topology determinant of membrane proteins, in addition to a hydrophobic segment and positive charges of the nascent chain.

ATP-binding cassette transporter isoform C2 localizes to the apical plasma membrane via interactions with scaffolding protein.

ATP-binding cassette transporter isoform C2 (ABCC2) localizes to the apical plasma membrane in polarized cells. Apical localization of ABCC2 in hepatocytes plays an important role in biliary excretion of endobiotics and xenobiotics, but the mechanism by which ABCC2 localizes to the apical membrane has not been conclusively elucidated. Here, we investigate the role of scaffolding proteins on ABCC2 localization with a focus on the function of PDZK1 (post-synaptic density 95/disk large/zonula occludens-1 domain containing 1) in regulating ABCC2 localization. The C-terminal 77 residues of ABCC2 were used to probe interacting proteins from HepG2 cells. Protein mass fingerprinting identified PDZK1 as a major interacting protein. PDZK1 associated with the plasma membrane, most likely at the apical vacuoles of HepG2 cells. Affinity pull-down assays confirmed that the C-terminal NSTKF of ABCC2 bound to the fourth PDZ domain of PDZK1. Removal of this PDZ-binding motif significantly reduced the normal apical localization of ABCC2. In HepG2 cells, overexpression of this fourth domain overcame endogenous PDZK1 and reduced the ABCC2 localization at the apical membrane with a reciprocal increase of intracellular accumulation of mislocalized ABCC2. These results suggest a possible role for an interaction between ABCC2 and PDZK1 in apical localization of ABCC2 in hepatocytes.

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.