N-terminal Cytoplasmic Domain Research Articles

Membrane-associated RING-CH (MARCH) 8 mediates the ubiquitination and lysosomal degradation of the transferrin receptor

The transferrin receptor (TfR) mediates the uptake of transferrin (Tf)-bound iron from the plasma into the cells of peripheral tissues. The TfR continuously recycles between the plasma membrane and early/recycling endosomes. TfR expression is tightly controlled by the intracellular iron concentration through the regulation of TfR mRNA stability. However, much less is known about the mechanism by which TfR is degraded in cells. Previously, we reported a correlation between TfR ubiquitination and its iron-induced lysosomal degradation. The identification and characterization of a specific ubiquitin ligase for TfR is important in understanding the mechanism of iron homeostasis. Here, we show that membrane-associated RING-CH (MARCH) 8 ubiquitinates TfR and promotes its lysosomal degradation. Similar to other RING-type ubiquitin ligases, the RING-CH domain of MARCH8, which is located in the N-terminal cytoplasmic domain, is essential for the ubiquitination and downregulation of TfR. MARCH8 specifically recognizes the transmembrane domain of TfR and mediates ubiquitination of its cytoplasmic domain. In addition, the six-amino-acid sequence located in the C-terminal domain of MARCH8, which is highly conserved among different species, is required for the downregulation of TfR. Finally, and most importantly, TfR expression was markedly increased by siRNA-mediated knockdown of endogenous MARCH8. These findings demonstrate that the endogenous level of MARCH8 regulates TfR protein turnover through the downregulation and ubiquitination of TfR.

A Cytoplasmic Interaction between Junctin and RyRs with Major Consequences for RyR1 and RyR2 Activity In Vitro

Junctin is an intrinsic sarcoplasmic reticulum (SR) membrane protein in cardiac and skeletal muscle. The bulk of the protein is within the lumen of the SR where it binds to ryanodine receptor (RyR) calcium release channels and regulates Ca2+ release from the SR. We have previously reported that junctin added to the luminal solution increases RyR1 activity in lipid bilayers [1].The contributions of the luminal and cytoplasmic domains of junctin to RyR activation have not previously been explored, although it is assumed that activation depends on interactions between their luminal domains. To explore this further, we expressed junctin's C-terminal domain (Cjun), synthesised a cytoplasmic N-terminal domain peptide (Njun) and isolated full length junctin (FLjun) from skeletal muscle. Consistent with previous findings [1,2], FLjun in luminal solutions activated purified RyR1 and RyR2. We predicted that luminal Cjun would similarly activate RyRs if the proteins interact only via their luminal domains. Unexpectedly, (a) luminal Cjun strongly inhibited RyR1 and RyR2 and (b) cytoplasmic Njun activated channels to a greater degree than luminal FLjun and also bound to cytoplasmic fragments of RyR1 [3]. Neither luminal Njun, nor scrambled Njun in cytoplasmic solution influenced channel activity. To explore cytoplasmic Njun and luminal Cjun effects further, we added both domains sequentially to channels. Excess activation by Njun was reduced to FLjun levels by adding Cjun, while significant inhibition by Cjun added first was reduced by Njun, it remained significantly different from the activation by FLjun. Therefore cytoplasmic interactions between junctin and RyRs determines the overall effect of junctin on channel activity.1. Wei et al. (2009) Int J Biochem Cell Biol41;2214.2. Gyorke et al. (2004) Biophys J 86:2121.3. Mirza et al. (2013) Biophysics abstract.

Phosphorylation of Chs2p regulates interaction with COPII

Trafficking of the chitin synthase Chs2p from the endoplasmic reticulum (ER) to the bud-neck in late mitosis is tightly regulated by the cell cycle via phosphorylation of serine residues in the N-terminus of the protein. Here, we describe the effects of Chs2p phosphorylation on the interaction with coat protein complex II (COPII). Identification of a cdc5(ts) mutant, which fails to transport Chs2p-3xGFP to the bud-neck and instead accumulates the protein in intracellular puncta, led us to discover that Chs2p-3xGFP accumulates at ER exit sites in metaphase-arrested wild-type cells. Using an in vitro ER vesicle formation assay we showed that phosphorylation of Chs2p by the cyclin-dependent kinase CDK1 prevents packaging into COPII vesicles, whereas dephosphorylation of Chs2p by the phosphatase Cdc14p stimulates selection into the vesicles. We found that the cytoplasmic N-terminal domain of Chs2p, which contains the CDK1 phosphorylation sites, interacts with the COPII component Sec24p in a yeast two-hybrid assay and that phosphomimetic substitutions of serines at the CDK1 consensus sites reduces the interaction. Our data suggest that dephosphorylation functions as a molecular switch for regulated ER exit of Chs2p.

Different Modes of Interaction between Junctin and Ryanodine Receptors

The sarcoplasmic reticulum (SR) Ca2+ store in skeletal and cardiac muscle is important in regulating Ca2+ release and the strength of contraction, through key proteins including the ryanodine receptor (RyR), calsequestrin (CSQ) and intrinsic membrane co-proteins, junctin and triadin, which link CSQ to RyRs. Junctin contributes to SR Ca2+ release, store size, CSQ2 depolymerisation and activates RyR1 and RyR2 in lipid bilayers when added to the luminal solution (1). Two regions of junctin's luminal domain interact with luminal loops on RyR2 (2), indicating that junctin modulates RyR2 from within the SR lumen. Similar experiments have not been done with RyR1 and cytoplasmic interactions between junctin and RyRs have not been fully explored in RyR1 or RyR2.To explore these interactions, we isolated full length junctin (FLjun) from skeletal muscle, expressed the C-terminal domain of junctin (Cjun) in E. coli and synthesised the Njun peptide corresponding to cytoplasmic N-terminal domain. Deletion constructs of cytoplasmic and luminal domains of RyR1 were expressed in HEK293 cells. Affinity chromatography and co-immunoprecipition confirmed that the FLjun binds to RyR1 and RyR2 and revealed that Njun binds to cytoplasmic regions of RyR1, while Cjun binds to two luminal loops on RyR1, one between TM5 and TM6 (aa4583-4626), and the second in a construct containing the pore helix and adjacent luminal residues (aa4861-4910). Results from lipid bilayers (Li, Mirza, Beard, Dulhunty abstract) indicate that junctin regulates RyR1 and RyR2 via cytoplasmic and luminal interactions and, unexpectedly, the cytoplasmic interaction is functionally dominant. Njun binding to the cytoplasmic domain of RyR1 must underly this action. We predict that Njun will also bind to the cytoplasmic domain of RyR2.1.Wei L et al. (2009) Int J Biochem Cell Bio 41, 2214.2.Altschafl et al. (2011) J Physiol 589, 6063.

Lipopolysaccharide induces CD38 expression and solubilization in J774 macrophage cells.

CD38, an ADP ribosyl cyclase, is a 45 kDa type II transmembrane protein having a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain, expressed on the surface of various cells including macrophages, lymphocytes, and pancreatic β cells. It is known to be involved in cell adhesion, signal transduction and calcium signaling. In addition to its transmembrane form, CD38 is detectable in biological fluids in soluble forms. The mechanism by which CD38 is solubilized from the plasma membrane is not yet clarified. In this study, we found that lipopolysaccharide (LPS) induced CD38 upregulation and its extracellular release in J774 macrophage cells. Furthermore, it also increased CD38 expression at the mRNA level by activating the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. However, LPS decreased the levels of CD38 in the plasma membrane by releasing CD38 into the culture supernatant. LPS-induced CD38 release was blocked by the metalloproteinase-9 inhibitor indicating that MMP-9 solubilizes CD38. In conclusion, the present findings demonstrate a potential mechanism by which C38 is solubilized from the plasma membrane.

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3

Band 3 (B3), the anion transporter, is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In this study, using a variety of experimental approaches including cross-linking studies, fluorescence and dichroic measurements, surface plasmon resonance analysis, and proteolytic digestion assays, we document that the antioxidant protein peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in the interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine of RBCs prevented membrane association of PRDX2, implying overlapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes, in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acids of this domain are crucial for PRDX2 membrane association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function.

The B. subtilis MgtE Magnesium Transporter Can Functionally Compensate TRPM7-Deficiency in Vertebrate B-Cells

Recent studies have shown that the vertebrate magnesium transporters Solute carrier family 41, members 1 and 2 (SLC41A1, SLC41A2) and Magnesium transporter subtype 1 (MagT1) can endow vertebrate B-cells lacking the ion-channel kinase Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) with a capacity to grow and proliferate. SLC41A1 and SLC41A2 display distant homology to the prokaryotic family of Mg2+ transporters, MgtE, first characterized in Bacillus subtilis. These sequence similarities prompted us to investigate whether MgtE could potentially compensate for the lack of TRPM7 in the vertebrate TRPM7-deficient DT40 B-cell model system. Here, we report that overexpression of MgtE is able to rescue the growth of TRPM7-KO DT40 B-cells. However, contrary to a previous report that describes regulation of MgtE channel gating by Mg2+ in a bacterial spheroplast model system, whole cell patch clamp analysis revealed no detectable current development in TRPM7-deficient cells expressing MgtE. In addition, we observed that MgtE expression is strongly downregulated at high magnesium concentrations, similar to what has been described for its vertebrate homolog, SLC41A1. We also show that the N-terminal cytoplasmic domain of MgtE is required for normal MgtE channel function, functionally confirming the predicted importance of this domain in regulation of MgtE-mediated Mg2+ entry. Overall, our findings show that consistent with its proposed function, Mg2+ uptake mediated by MgtE is able to restore cell growth and proliferation of TRPM7-deficient cells and supports the concept of functional homology between MgtE and its vertebrate homologs.

Optical Control of Endogenous Proteins with a Photoswitchable Conditional Subunit Reveals a Role for TREK1 in GABAB Signaling

Selective ligands are lacking for many neuronal signaling proteins. Photoswitched tethered ligands (PTLs) have enabled fast and reversible control of specific proteins containing a PTL anchoring site and have been used to remote control overexpressed proteins. We report here a scheme for optical remote control of native proteins usinga "photoswitchable conditional subunit" (PCS), which contains the PTL anchoring site as well as a mutation that prevents it from reaching the plasma membrane. In cells lacking native subunits for the protein, the PCS remains nonfunctional internally. However, in cells expressing native subunits, the native subunit and PCS coassemble, traffic to the plasma membrane, and place the native protein under optical control provided by the coassembled PCS. We apply this approach to the TREK1 potassium channel, which lacks selective, reversible blockers. We find that TREK1, typically considered to be a leak channel, contributes to the hippocampal GABA(B) response.

Interaction of Voltage-gated Sodium Channel Nav1.6 (SCN8A) with Microtubule-associated Protein Map1b

The mechanism by which voltage-gated sodium channels are trafficked to the surface of neurons is not well understood. Our previous work implicated the cytoplasmic N terminus of the sodium channel Na(v)1.6 in this process. We report that the N terminus plus the first transmembrane segment (residues 1-153) is sufficient to direct a reporter to the cell surface. To identify proteins that interact with the 117-residue N-terminal domain, we carried out a yeast two-hybrid screen of a mouse brain cDNA library. Three clones containing overlapping portions of the light chain of microtubule-associated protein Map1b (Mtap1b) were recovered from the screen. Interaction between endogenous Na(v)1.6 channels and Map1b in mouse brain was confirmed by co-immunoprecipitation. Map1b did not interact with the N terminus of the related channel Na(v)1.1. Alanine-scanning mutagenesis of the Na(v)1.6 N terminus demonstrated that residues 77-80 (VAVP) contribute to interaction with Map1b. Co-expression of Na(v)1.6 with Map1b in neuronal cell line ND7/23 resulted in a 50% increase in current density, demonstrating a functional role for this interaction. Mutation of the Map1b binding site of Na(v)1.6 prevented generation of sodium current in transfected cells. The data indicate that Map1b facilitates trafficking of Na(v)1.6 to the neuronal cell surface.

Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system.

The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are required for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.

Structural basis for the regulation of L-type voltage-gated calcium channels: interactions between the N-terminal cytoplasmic domain and Ca2+-calmodulin

It is well-known that the opening of L-type voltage-gated calcium channels can be regulated by calmodulin (CaM). One of the main regulatory mechanisms is calcium-dependent inactivation (CDI), where binding of apo-CaM to the cytoplasmic C-terminal domain of the channel can effectively sense an increase in the local calcium ion concentration. Calcium-bound CaM can bind to the IQ-motif region of the C-terminal region and block the calcium channel, thereby providing a negative feedback mechanism that prevents the rise of cellular calcium concentrations over physiological limits. Recently, an additional Ca2+/CaM-binding motif (NSCaTE, N-terminal spatial Ca2+ transforming element) was identified in the amino terminal cytoplasmic region of Cav1.2 and Cav1.3. This motif exists only in Cav1.2 and Cav1.3 channels, and a pronounced N-lobe (Ca2+/CaM) CDI effect was found for Cav1.3. To understand the molecular basis of this interaction, the complexes of Ca2+/CaM with the biosynthetically produced N-terminal region (residues 1–68) and NSCaTE peptide (residues 48–68) were investigated. We discovered that the NSCaTE motif in the N-terminal cytoplasmic region adopts an α-helical conformation, most likely due to its high alanine content. Additionally, the complex exhibits an unusual 1:2 protein:peptide stoichiometry when bound to Ca2+-CaM, and the N-lobe of CaM has a much stronger affinity for the peptide than the C-lobe. The complex structures of the isolated N- and C-lobe of Ca2+/CaM and the NSCaTE peptide were determined by nuclear magnetic resonance spectroscopy and data-driven protein-docking methods. Moreover, we also demonstrated that calcium binding protein 1, which competes with CaM for binding to the C-terminal cytoplasmic domain, binds only weakly to the NSCaTE region. The structures provide insights into the possible roles of this motif in the calcium regulatory network. Our study provides structural evidence for the CaM-bridge model proposed in previous studies.

Role of the Cytoplasmic N-Terminal Domain of the Cardiac Sodium Channel Alpha-Subunit

Heterozygous loss-of-function mutations in the SCN5A gene encoding the cardiac Na+ channel Nav1.5 cause various hereditary arrhythmias including Brugada syndrome (BrS), a disease characterized by ST-segment elevation in the ECG right precordial leads, and an increased risk of sudden cardiac death. Here, we aimed to study the role of the Nav1.5 evolutionarily conserved N-terminus of which the function remains unknown. We characterized two mutations within the cytoplasmic N-terminus of Nav1.5, R104W and R121W identified in BrS patients and a construct where this region was deleted.

Bimodal Regulation of hERG Gating by the N-Terminal Tail Revealed by Voltage Clamp Fluorometry

The cytoplasmic N-terminal domain of human-ether-a-go-go related gene (hERG) K+ channels contains an amphipathic α-helix and an unstructured tail. Deletion of residues 2-9 (Δ2-9, the unstructured tail) or 2-25 (Δ2-25, α-helix and unstructured tail) both accelerated deactivation and slowed the rate of activation. However, while Δ2-9 shifted the V0.5 of activation in the depolarized direction, Δ2-25 did not, suggesting differential effects of the two deletions on the activation process.In this study we have used voltage clamp fluorometry to investigate the mechanism of how these N-terminal deletions affect voltage sensor movement during the activation process. The V0.5 of the fluorescence-voltage relationship for Δ2-9 was shifted to more depolarized potentials (18.2 ± 1.5 mV, n= 9) compared to control (0 ± 1.8 mV, n=12, ANOVA) indicating that Δ2-9 affects activation gating by shifting the voltage range over which the voltage sensor moves, while the V0.5 of activation for Δ2-25 (1 ± 2.9 mV, n=7) was not significantly different from control. In contrast, both Δ2-9 and Δ2-25 have slow rates of current activation with no observable effect on the fluorescent report of the rate of voltage sensor movement, suggesting that both Δ2-9 and Δ2-25 slowed rates of activation independent of the voltage sensor.Our data therefore suggests a bimodal regulation of activation gating by the N-terminal domain of hERG channels. We propose that the α-helical region modulates activation gating via an effect on movement of the voltage sensor while the N-terminal tail (residues 1-9) interacts with the cytoplasmic activation gate to regulate the rates of pore opening and closing.

Modeling IBD in the mouse through random germline mutagenesis

Inflammatory bowel diseases (IBD), including both ulcerative colitis and Crohn's disease, have strong genetic components. In mice, several mutations are known to favor IBD or to inhibit intestinal inflammation. But a comprehensive picture of the pathogenesis of IBD cannot be assembled based on the limited information so far available from genetic analyses. To unveil new genes important for the pathogenesis of IBD, we took an unbiased, hypothesis-free view of IBD, using germline mutagenesis to analyze the phenomenon in mice. We screen for mutations that cause susceptibility to IBD on a defined genetic background using the Dextran sodium sulfate (DSS) colitis model, positionally clone these mutations, and form hypotheses concerning the participation of each validated gene in the disease pathogenesis. A total of 5846 mice have been screened by placing them on 1% DSS in drinking water for a period of 7 days. During this time period all animals are weighed daily, and extreme outliers are retrieved for further study. A total of 16 transmissible mutations causing hypersensitivity to DSS have been detected. Of these, 6 have been identified, and fall into 5 genes. Failure of epithelial integrity is sensed by Toll-like receptors (TLR) on epithelial cells. Wound closure also additionally requires water transport via aquaporin 3, signaling via the EGF receptor pathway, and an intact unfolded protein response, which depends on Mbtps1, also known as the site-1 protease. Relatively common mutations affecting the structure of Mucin-2 also enhance susceptibility to colitis, evidently by increasing ER stress. In addition, genes with unknown functions have been identified causing increased susceptibility to DSS colitis. One of the mutant mice identified in this screen was homozygous for a mutation in an uncharacterized gene. The gene is predicted to encode a protein with an unstructured cytoplasmic N-terminal domain and a C-terminal domain containing five transmembrane α helices connected by short loops. The mutation lies in the splice acceptor site of intron 3 of the seven exon gene, and affects a thymine base five nucleotides from the next exon. cDNA sequencing revealed that the mutation results in skipping of exon 4. Splicing of exon 3 to exon 5 creates a frameshift and a premature stop codon. The Klein-Zschocher mouse showed severe weight loss after administration of 1% DSS in the drinking water. Periodic acid-Schiff stain staining of small intestines and colons of Klein-Zschocher mice revealed reduced numbers of Paneth and goblet cells in naïve mutant mice. Furthermore, electron microscopy of the small intestine and colon of naive Klein-Zschocher mice showed abnormal vesicles in Paneth and goblet cells of the mutant mice. Defective Paneth cells of Klein-Zschocher mutant mice show enhanced autophagy as demonstrated by LC3 Western blotting. In addition, the intestinal permeability was increased 85% in Klein-Zschocher mice demonstrating epithelial barrier dysfunction in these mice. Disturbed epithelial integrity together with abnormal Paneth and goblet cells explain the increased susceptibility in the DSS colitis model. Taken together, using random mutagenesis and the DSS mouse model we found new genes and new functions of known genes that might play a role in IBD. A coherent picture of the essential events required for homeostasis has begun to emerge.

Abstract 17271: An Alternatively Spliced Kcnh2 Variant Restores Cardiac Repolarization in a Model of Long QT Syndrome: Implications for Phenotypic Variation in Human Long QT Syndrome

Introduction: Mutations in KCNH2 ( HERG1 , LQT-2) are a common cause of Long QT Syndrome (LQTS). Despite sharing the same mutation, affected family members often display marked differences in disease severity, including risk of sudden cardiac death. The genetic basis of this phenotypic variation is not known. We previously validated a zebrafish model of human LQT-2. Here, we use a repolarization-deficient zebrafish mutant ( breakdance , bre ) to probe the molecular basis of phenotypic variation. Methods: Bre is due to a recessive kchn2 mutation (I59S) within the cytoplasmic N-terminal domain. 5’-RACE and qPCR were used to clone and quantify novel N-terminal variants from zebrafish and human heart. Biophysical properties of zebrafish and human variants were characterized using voltage- and patch-clamp techniques. In vivo functional effects were determined by over-expressing the variants in knch2-null zebrafish embryos, and assaying for ability to restore ventricular repolarization by characterizing phenotypes of the embryonic heart at 48 hour-post-fertilization. Results: We identified a novel, alternatively spliced N-terminal variant, kcnh2 1c , with in-frame deletion of Exon 2-6. The 1c variant constitutes ~40% of the kcnh2 transcript, and is expressed 4X greater in atrium than ventricle. The 1c variant deletes the disease-causing mutation, I59S. Activation of kcnh2 1c channels is similar to WT, but deactivation is accelerated. In vivo , kcnh2 1c functions similar to WT, and is able to partially restore ventricular repolarization in knch2-null zebrafish. We identified 2 novel N-terminal variants in human heart that are also differentially expressed between the atrium and ventricle. Biophysical function of the human variants is similar to zebrafish 1c. Conclusion: Kcnh2 1c modulates cardiac repolarization in the bre mutant by excising the disease-causing mutation. Approximately 30% of LQT-2 mutations localize to the N-terminus, and are subject to alternative splicing. By excising N-terminal mutations human 1c variants may modulate repolarization and influence phenotypic expression in LQTS.

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.

Identification of Sorting Motifs of AtβFruct4 for Trafficking from the ER to the Vacuole Through the Golgi and PVC

Although much is known about the molecular mechanisms involved in transporting soluble proteins to the central vacuole, the mechanisms governing the trafficking of membrane proteins remain largely unknown. In this study, we investigated the mechanism involved in targeting the membrane protein, AtβFructosidase 4 (AtβFruct4), to the central vacuole in protoplasts. AtβFruct4 as a green fluorescent protein (GFP) fusion protein was transported as a membrane protein during transit from the endoplasmic reticulum (ER) through the Golgi apparatus and the prevacuolar compartment (PVC). The N-terminal cytosolic domain of AtβFruct4 was sufficient for transport from the ER to the central vacuole and contained sequence motifs required for trafficking. The sequence motifs, LL and PI, were found to be critical for ER exit, while the EEE and LCPYTRL sequence motifs played roles in trafficking primarily from the trans Golgi network (TGN) to the PVC and from the PVC to the central vacuole, respectively. In addition, actin filaments and AtRabF2a, a Rab GTPase, played critical roles in vacuolar trafficking at the TGN and PVC, respectively. On the basis of these results, we propose that the vacuolar trafficking of AtβFruct4 depends on multiple sequence motifs located at the N-terminal cytoplasmic domain that function as exit and/or sorting signals in different stages during the trafficking process.

Definition of Membrane Topology and Identification of Residues Important for Transport in Subunit a of the Vacuolar ATPase

Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7.

SLC41A1 Mg2+ transport is regulated via Mg2+-dependent endosomal recycling through its N-terminal cytoplasmic domain

SLC41A1 (solute carrier family 41, member A1) is a recently described vertebrate member of the MgtE family of Mg(2+) transporters. Although MgtE transporters are found in both prokaryotic and eukaryotic organisms, and are highly conserved, little is known about the regulation of their Mg(2+) transport function. In the present study, we have shown that endogenous SLC41A1 transporter expression is post-transcriptionally regulated by extracellular Mg(2+) in TRPM7 (transient receptor potential cation channel, subfamily M, member 7)-deficient cells, suggesting that SLC41A1 transporters underlie a novel plasma membrane Mg(2+) transport function. Consistent with this conclusion, structure-function analyses of heterologous SLC41A1 transporter expression demonstrate that SLC41A1 transporters exhibit the same plasma membrane orientation as homologous bacterial MgtE proteins, are capable of complementing growth of TRPM7-deficient cells only when the Mg(2+) transporting pore is intact, and require an N-terminal cytoplasmic domain for Mg(2+)-dependent regulation of lysosomal degradation and surface expression. Taken together, our results indicate that SLC41A1 proteins are a central component of vertebrate Mg(2+) transport systems, and that their Mg(2+) transport function is regulated primarily through an endosomal recycling mechanism involving the SLC41A1 N-terminal cytoplasmic domain.

Essential Role of Endocytosis of the Type II Transmembrane Serine Protease TMPRSS6 in Regulating Its Functionality

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.