N-terminal Cytosolic Domain Research Articles

Alternative Splicing of the N-Terminal Cytosolic and Transmembrane Domains of P2X7 Controls Gating of the Ion Channel by ADP-Ribosylation

P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD+-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD+ and ATP.

Lipid interaction of the C terminus and association of the transmembrane segments facilitate atlastin-mediated homotypic endoplasmic reticulum fusion

The homotypic fusion of endoplasmic reticulum (ER) membranes is mediated by atlastin (ATL), which consists of an N-terminal cytosolic domain containing a GTPase module and a three-helix bundle followed by two transmembrane (TM) segments and a C-terminal tail (CT). Fusion depends on a GTP hydrolysis-induced conformational change in the cytosolic domain. Here, we show that the CT and TM segments also are required for efficient fusion and provide insight into their mechanistic roles. The essential feature of the CT is a conserved amphipathic helix. A synthetic peptide corresponding to the helix, but not to unrelated amphipathic helices, can act in trans to restore the fusion activity of tailless ATL. The CT promotes vesicle fusion by interacting directly with and perturbing the lipid bilayer without causing significant lysis. The TM segments do not serve as mere membrane anchors for the cytosolic domain but rather mediate the formation of ATL oligomers. Point mutations in either the C-terminal helix or the TMs impair ATL's ability to generate and maintain ER morphology in vivo. Our results suggest that protein-lipid and protein-protein interactions within the membrane cooperate with the conformational change of the cytosolic domain to achieve homotypic ER membrane fusion.

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.

NMR structure note: the ferrous iron transport protein C (FeoC) from Klebsiella pneumoniae

Iron is an essential nutrient involved in many important biological processes ranging from electron transfer, oxygen transport, gene regulation, photosynthesis, N2 fixation to DNA biosynthesis (Andrews et al. 2003). Thus, the ability to acquire sufficient quantities of iron from the environment is vital for the survival of living organisms. This is particularly true for pathogens that must compete with the host iron-withdrawal response for their iron supplies. However, iron is also toxic and excess iron can be detrimental for the living system (Touati 2000). It is, therefore, essential for almost all life forms to maintain proper iron homeostasis (Andrews et al. 2003). Feo is a unique ferrous iron transporter commonly utilized by bacterial systems for acquiring ferrous iron from the environment (Cartron et al. 2006). It was first discovered in Escherichia coli K-12 (Hantke 1987). Subsequently the feo locus was cloned and sequenced (Kammler et al. 1993). The importance of the Feo system has been confirmed in several systems. Both E. coli and Salmonella feoB mutants are attenuated in their abilities to colonize the mouse intestine presumably due to their inability to transport ferrous iron within the anaerobic environment of the mouse intestine (Stojiljkovic et al. 1993; Tsolis et al. 1996). In H. pylori, FeoB appears to provide the main route of iron uptake. It is required for H. pylori colonization of mouse gastric mucosa, as well as for normal growth and iron-uptake under iron restricted conditions (Velayudhan et al. 2000). FeoB is also required for intracellular growth of Legionella pneumophila (Robey and Cianciotto 2002). Thus, various studies have clearly established an in vivo role for Feo in colonization of the gut and in virulence. The feo operon of enterobacteria encodes three proteins: FeoA, FeoB and FeoC. FeoA is a small SH3-like protein predicted to act as GTPase activating protein (GAP) and/or Fe-dependent repressor (Cartron et al. 2006). FeoB is a large protein (773 a.a.) composing of a 270-residue cytosolic N-terminal domain (NFeoB, residues 1–270) that contains a G-protein domain (a.a. 1–170) and a helical bundle S-domain (a.a. 171–270) presumed to be a GDP-dissociation inhibitor (GDI). The C-terminal region of FeoB is a helical transmembrane domain which is likely to act as the ferrous iron permease and essential for ferrous iron transport activity. FeoC (also called yhgG) is a small 78-residue, hydrophilic winged-helix domain (WHD) found only in c-proteobacteria. The feoC gene was found preceding the feoB gene in the feo operon. Multiple sequence alignment of FeoC proteins shows that they possess four conserved cysteine residues (CxxGxCKxCPx4-7C) that are likely to provide a binding site for an [Fe–S]-cluster. Thus, FeoC is presumed to be a [Fe–S]dependent transcriptional regulator directly controlling the expression of the feo operon (Cartron et al. 2006). Given its importance and novelty, there has had increasing interest in dissecting the molecular basis of the Feo system. In particular, the presence of a G-protein motif in the intracellular domain of FeoB (NFeoB) has spurred considerable interest in understanding its molecular basis and the structures of NFeoB from several species have K.-W. Hung T. Juan Y. Hsu T. H. Huang (&) Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC e-mail: bmthh@ibms.sinica.edu.tw

Solution structure of subunit a, a 104-363, of the Saccharomyces cerevisiae V-ATPase and the importance of its C-terminus in structure formation

The 95 kDa subunit a of eukaryotic V-ATPases consists of a C-terminal, ion-translocating part and an N-terminal cytosolic domain. The latter's N-terminal domain (~40 kDa) is described to bind in an acidification-dependent manner with cytohesin-2 (ARNO), giving the V-ATPase the putative function as pH-sensing receptor. Recently, the solution structure of the very N-terminal segment of the cytosolic N-terminal domain has been solved. Here we produced the N-terminal truncated form SCa₁₀₄₋₃₆₃ of the N-terminal domain (SCa₁₋₃₆₃) of the Saccharomyces cerevisiae V-ATPase and determined its low resolution solution structure, derived from SAXS data. SCa₁₀₄₋₃₆₃ shows an extended S-like conformation with a width of about 3.88 nm and a length of 11.4 nm. The structure has been superimposed into the 3D reconstruction of the related A₁A₀ ATP synthase from Pyrococcus furiosus, revealing that the SCa₁₀₄₋₃₆₃ fits well into the density of the collar structure of the enzyme complex. To understand the importance of the C-terminus of the protein SCa₁₋₃₆₃, and to determine the localization of the N- and C-termini in SCa₁₀₄₋₃₆₃, the C-terminal truncated form SCa₁₀₆₋₃₂₄ was produced and analyzed by SAXS. Comparison of the SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ shapes showed that the additional loop region in SCa₁₀₄₋₃₆₃ consists of the C-terminal residues. Whereas SCa₁₀₄₋₃₆₃ is monomeric in solution, SCa₁₀₆₋₃₂₄ forms a dimer, indicating the importance of the very C-terminus in structure formation. Finally, the solution structure of SCa₁₀₄₋₃₆₃ and SCa₁₀₆₋₃₂₄ will be discussed in terms of the topological arrangement of subunit a and cytoheisn-2 in V-ATPases.

Toward Unraveling the Structure and Molecular Basis of Ferrous Iron Transport of Feo from Klebsiella Pneumoniae

Feo from K. pneumoniae is a unique type of ferrous iron transporter for bacteria. It consists of three proteins, FeoA, a small SH3-domain like protein; FeoB, a large protein consisting of a cytosolic N-terminal G-protein domain and a C-terminal transmembrane domain that may serve as a Fe++ permease; and FeoC, a small putative transcriptional factor. Using X-ray crystallography and NMR we have solved the structure of FeoA, the intracellular domain of FeoB (NFeoB), FeoC and NFeoB/FeoC complex. In the structures, a canonical G-protein domain (G domain) is followed by a helical bundle domain (S-domain), which despite its lack of sequence similarity between species is structurally conserved.

XPORT-Dependent Transport of TRP and Rhodopsin

TRP channels have emerged as key biological sensors in vision, taste, olfaction, hearing, and touch. Despite their importance, virtually nothing is known about the folding and transport of TRP channels during biosynthesis. Here, we identify XPORT (exit protein of rhodopsin and TRP) as a critical chaperone for TRP and its G protein-coupled receptor (GPCR), rhodopsin (Rh1). XPORT is a resident ER and secretory pathway protein that interacts with TRP and Rh1, as well as with Hsp27 and Hsp90. XPORT promotes the targeting of TRP to the membrane in Drosophila S2 cells, a finding that provides a critical first step toward solving a longstanding problem inthe successful heterologous expression of TRP. Mutations in xport result in defective transport of TRP and Rh1, leading to retinal degeneration. Our results identify XPORT as a molecular chaperone and provide a mechanistic link between TRP channels and their GPCRs during biosynthesis and transport.

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.

Crystal Structure of the Cytoplasmic N-Terminal Domain of Subunit I, a Homolog of Subunit a, of V-ATPase

Subunit “a” is associated with the membrane-bound (V O) complex of eukaryotic vacuolar H +-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit “I” homolog of subunit a. The structure is composed of a curved long central α-helix bundle capped on both ends by two lobes with similar α/β architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H +-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

Mechanism of tumor resistance to cisplatin mediated by the copper transporter ATP7BThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

The Wilson disease protein (ATP7B) is a copper-transporting ATPase that is responsible for regulating copper homeostasis in human tissues. ATP7B is associated with cancer resistance to cisplatin, one of the most widely used anticancer drugs. This minireview discusses the possible mechanisms of tumor resistance to cisplatin mediated by ATP7B. Cisplatin binds to the N-terminal cytosolic domain of ATP7B, which contains multiple copper-binding sites. Active platinum efflux catalyzed by ATP7B is unlikely to significantly contribute to cisplatin resistance in vivo. Transient platinum sequestration in the metal-binding domain followed by transfer to an acceptor protein or a low molecular weight compound is proposed as an alternative mechanism of cisplatin detoxification in the cell.

The N-terminal domain of Orai3 determines selectivity for activation of the store-independent ARC channel by arachidonic acid

Although highly selective Ca2+ entry pathways play a critical role in agonist-activated Ca2+ signals in non-excitable cells, only with the recent discovery of the Orai proteins have the first insights into the molecular nature of these pathways been possible. To date, just two such highly Ca2+-selective "Orai channels" have been identified in native cells - the store-operated CRAC channels and the store-independent, arachidonic acid-activated ARC channels. Studies have shown that the functional CRAC channel pore is formed by a tetrameric arrangement of Orai1 subunits, whilst a heteropentamer of three Orai1 subunits and two Orai3 subunits forms the functional ARC channel pore. Importantly, this inclusion of Orai3 subunits in the ARC channel structure has been shown to play a specific role in determining the selectivity of these channels for activation by arachidonic acid. Using an approach based on the expression of various concatenated constructs, we examined the basis for this Orai3-dependent effect on selectivity for arachidonic acid. We show that, whilst heteropentamers containing only one Orai3 subunit are sensitive to arachidonic acid, specific selectivity for activation by this fatty acid is only achieved on inclusion of the second Orai3 subunit in the pentamer. Further studies identified the cytosolic N-terminal domain of Orai3 as the region specifically responsible for this switch in selectivity. Substitution of just this domain into an otherwise complete single Orai1 subunit within a concatenated 31111 pentamer is sufficient to change the resulting channel from one that is predominantly store-operated, to one that is exclusively activated by arachidonic acid.

Heme and non-heme iron transporters in non-polarized and polarized cells

BackgroundHeme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs), and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs.ResultsIn this study, we analyzed the subcellular localizations of 2 isoenzymes of HOs, 4 isoforms of divalent metal transporter 1 (DMT1), and 2 candidate heme transporters--heme carrier protein 1 (HCP1) and heme responsive gene-1 (HRG-1)--in non-polarized and polarized cells. In non-polarized cells, HCP1, HRG-1, and DMT1A-I are located in the plasma membrane. In polarized cells, they show distinct localizations: HCP1 and DMT1A-I are located in the apical membrane, whereas HRG-1 is located in the basolateral membrane and lysosome. 16Leu at DMT1A-I N-terminal cytosolic domain was found to be crucial for plasma membrane localization. HOs are located in smooth endoplasmic reticulum and colocalize with NADPH-cytochrome P450 reductase.ConclusionsHCP1 and DMT1A-I are localized to the apical membrane, and HRG-1 to the basolateral membrane and lysosome. These findings suggest that HCP1 and DMT1A-I have functions in the uptake of dietary heme and non-heme iron. HRG-1 can transport endocytosed heme from the lysosome into the cytosol. These localization studies support a model in which cytosolic heme can be degraded by HOs, and the resulting iron is exported into tissue fluids via the iron transporter ferroportin 1, which is expressed in the basolateral membrane in enterocytes or in the plasma membrane in macrophages. The liberated iron is transported by transferrin and reutilized for hemoglobin synthesis in the erythroid system.

Amino Acid Signaling in Yeast: Post-genome Duplication Divergence of the Stp1 and Stp2 Transcription Factors

When yeast cells detect external amino acids via their permease-like Ssy1 sensor, the cytosolic precursor forms of Stp1 and Stp2 transcription factors are activated by endoproteolytic removal of their N-terminal domains, a reaction catalyzed by the Ssy5 endoprotease. The processed Stp factors then migrate into the nucleus, where they activate transcription of several amino acid permease genes including AGP1. We report here that the STP1 and STP2 genes most likely derive from the whole genome duplication that occurred in a yeast ancestor. Although Stp1 and Stp2 have been considered redundant, we provide evidence that they functionally diverged during evolution. Stp2 is the only factor processed when amino acids are present at low concentration, and the transcriptional activation of AGP1 promoted by Stp2 is moderate. Furthermore, only Stp2 can sustain Agp1-dependent utilization of amino acids at low concentration. In contrast, Stp1 is only processed when amino acids are present at high concentration, and it promotes higher level transcriptional activation of AGP1. Domain swapping experiments show that the N-terminal domains of Stp1 and Stp2 are responsible for these proteins being cleaved at different amino acid concentrations. Last, induction of the DIP5 permease gene by amino acids depends on Stp2 but not Stp1. We propose that post-whole genome duplication co-conservation of the STP1 and STP2 genes was favored by functional divergence of their products, likely conferring to cells an increased ability to adapt to various amino acid supply conditions.

Orai3 and the Selective Activation of the Arc Channel by Arachidonic Acid

The store-operated CRAC channels and the store-independent, arachidonic acid-activated ARC channels represent the founding members of a new family of biophysically similar, highly Ca2+-selective, Ca2+ entry channels - the “Orai channels”. Both of these channels are dependent on STIM1 for their activation, but they differ in the pool of STIM1 responsible. Thus, whereas STIM1 in the ER regulates the CRAC channels on store-depletion, ARC channels are exclusively regulated by the pool of STIM1 that constitutively resides in the PM.Recent studies have shown that the functional CRAC channel pore is formed by a tetrameric arrangement of Orai1 units. In contrast, a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits forms the functional ARC channel pore (Mignen et al. J. Physiol. 587: 4181). Importantly, this inclusion of Orai3 subunits in the channel structure has been shown to play a specific, and unique, role in determining the selectivity of the ARC channels for activation by arachidonic acid. Using an approach based on the generation and expression of various concatenated constructs, we examined the basis for this Orai3-dependent effect on selectivity for arachidonic acid. These studies revealed that, whilst heteropentamers containing only one Orai3 subunit are sensitive to arachidonic acid, specific selectivity for activation by this fatty acid is only achieved on inclusion of the second Orai3 subunit in the pentamer. Further studies identified the cytosolic N-terminal domain as the region of the Orai3 molecule that is specifically responsible for this switch in selectivity. Substitution of just this domain into an otherwise complete Orai1 subunit within a concatenated 31111 pentamer is sufficient to change the resulting channel from one that is predominantly store-operated, to one that is essentially exclusively activated by arachidonic acid.

The enigmatic role of Mim1 in mitochondrial biogenesis

The translocase of the outer mitochondrial membrane (TOM complex) is a multi-subunit complex that serves as the general entry site for newly synthesized proteins into the organelle. The assembly of this complex is a multi-step process that requires the coordinated action of several proteins. A central, but rather undefined role in this process is played by Mim1, a mitochondrial outer membrane protein. The deletion of MIM1 leads to severe defects in the biogenesis of TOM complex subunits and to altered mitochondrial morphology. The protein is built from an N-terminal cytosolic domain, a central transmembrane segment, and a C-terminal domain facing the intermembrane space. In this review we summarize our current knowledge on the structure–function relationship of Mim1 and discuss some possibilities for its molecular function.

Insights into the effect of detergents on the full-length rhomboid protease from Pseudomonas aeruginosa and its cytosolic domain

Rhomboids comprise a family of intramembrane serine proteases that catalyze the cleavage of transmembrane segments within the lipid membrane to achieve a wide range of biological functions. A subset of bacterial rhomboids possesses an N-terminal cytosolic domain that appears to enhance proteolytic activity via an unknown mechanism. Structural analysis of a full-length rhomboid would provide new insights into this mechanism, an objective that solution NMR has the potential to realize. For this purpose we purified the rhomboid from Pseudomonas aeruginosa in a range of membrane-mimetic media, evaluated its functional status in vitro and investigated the NMR spectroscopic properties of these samples. In general, NMR signals could only be observed from the cytosolic domain, and only in detergents that did not support rhomboid activity. In contrast, media that supported rhomboid function did not show these resonances, suggesting an association between the cytosolic domain and the protein–detergent complex. Investigations into the ability of the isolated cytosolic domain to bind detergent micelles revealed a denaturing interaction, whereas no interaction occurred with micelles that supported rhomboid activity. The cytosolic domain also did not show any tendency to interact with lipid bilayers found in small bicelles or vesicles made from Escherichia coli phospholipid extracts. Based on these data we propose that the cytosolic domain does not interact with the lipid membrane, but instead enhances rhomboid activity through interactions with some other part of the rhomboid, such as the catalytic core domain.

Structural Basis of Novel Interactions Between the Small-GTPase and GDI-like Domains in Prokaryotic FeoB Iron Transporter

The FeoB family proteins are widely distributed prokaryotic membrane proteins involved in Fe(2+) uptake. FeoB consists of N-terminal cytosolic and C-terminal transmembrane domains. The N-terminal region of the cytosolic domain is homologous to small GTPase (G) proteins and is considered to regulate Fe(2+) uptake. The spacer region connecting the G and TM domains reportedly functions as a GDP dissociation inhibitor (GDI)-like domain that stabilizes the GDP-binding state. However, the function of the G and GDI-like domains in iron uptake remains unclear. Here, we report the structural and functional analyses of the FeoB cytosolic domain from Thermotoga maritima. The structure-based mutational analysis indicated that the interaction between the G and GDI-like domains is important for both the GDI and Fe(2+) uptake activities. On the basis of these results, we propose a regulatory mechanism of Fe(2+) uptake.

NMR assignments of a 48 kDa tetramer of the T1 domain of the mammalian voltage gated potassium channel Kv1.4

The N-terminal cytosolic T1 domain of the mammalian voltage gated potassium channel Kv1.4 is strongly involved in the tetramerization of the Kv1.4 subunit that is required for forming a functional ion channel. The T1 domain forms a stable tetramer of 48 kDa in solution that cannot be dissociated into monomers. In spite of the high molecular mass it was possible to completely assign the backbone and part of the side chain resonances by multidimensional NMR spectroscopy on uniformly (2)H, (13)C, (15)N enriched protein. The secondary structure analysis derived from the chemical shifts is in line with the expectations from X-ray structures of related proteins.

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na(+)/H(+) exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.

Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase

Disassembly of the yeast V-ATPase into cytosolic V(1) and membrane V(0) sectors inactivates MgATPase activity of the V(1)-ATPase. This inactivation requires the V(1) H subunit (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767), but its mechanism is not fully understood. The H subunit has two domains. Interactions of each domain with V(1) and V(0) subunits were identified by two-hybrid assay. The B subunit of the V(1) catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V(1) subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V(0) subunit Vph1p. V(1)-ATPase complexes from yeast expressing H-NT are partially inhibited, exhibiting 26% the MgATPase activity of complexes with no H subunit. The H-CT domain does not copurify with V(1) when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V(1) lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V(1) was more stable than binding of either H-NT or H-CT, suggesting that both domains contribute to binding and inhibition. Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but this fragment of Vph1p does not bind to V(1) complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V(1)-ATPase activity and precludes V(0) interactions.