TM2 but not TM4 of subunit c'' interacts with TM7 of subunit a of the yeast V-ATPase as defined by disulfide-mediated cross-linking

The vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c') relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c'. Subunit c' contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu145 and Leu147 (Kawasaki-Nishi et al. (2003) J. Biol. Chem. 278, 41908-41913). We have now analyzed interaction of subunits a and c' using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c' centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c'. By contrast, TM4 of subunit c' does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other.     

Analysis of the membrane topology of transmembrane segments in the C-terminal hydrophobic domain of the yeast vacuolar ATPase subunit a (Vph1p) by chemical modification

The integral V(0) domain of the vacuolar (H(+))-ATPases (V-ATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100-kDa integral subunit of V(0) that plays an essential role in proton translocation. To better define the membrane topology of subunit a, unique cysteine residues were introduced into a Cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEG-mal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residues 602 and 620 and between residues 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734, and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7, and 8.     

Function and subunit interactions of the N-terminal domain of subunit a (Vph1p) of the yeast V-ATPase

The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V(0) domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V(0) domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V(0), suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V(0), factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO3 and MgATP did not, however, lead to an increase in passive proton conductance by free V(0), suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V(0). Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347-369) is important in anchoring the peripheral stator in V1V0.     

Incorporation of transmembrane peptides from the vacuolar H(+)-ATPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarized infrared spectroscopy

Peptides were designed that are based on candidate transmembrane sequences of the V o-sector from the vacuolar H (+)-ATPase of Saccharomyces cerevisiae. Spin-label EPR studies of lipid-protein interactions were used to characterize the state of oligomerization, and polarized IR spectroscopy was used to determine the secondary structure and orientation, of these peptides in lipid bilayer membranes. Peptides corresponding to the second and fourth transmembrane domains (TM2 and TM4) of proteolipid subunit c (Vma3p) and of the putative seventh transmembrane domain (TM7) of subunit a (Vph1p) are wholly, or predominantly, alpha-helical in membranes of dioleoyl phosphatidylcholine. All three peptides self-assemble into oligomers of different sizes, in which the helices are differently inclined with respect to the membrane normal. The coassembly of rotor (Vma3p TM4) and stator (Vph1p TM7) peptides, which respectively contain the glutamate and arginine residues essential to proton transport by the rotary ATPase mechanism, is demonstrated from changes in the lipid interaction stoichiometry and helix orientation. Concanamycin, a potent V-ATPase inhibitor, and a 5-(2-indolyl)-2,4-pentadienoyl inhibitor that exhibits selectivity for the osteoclast subtype, interact with the membrane-incorporated Vma3p TM4 peptide, as evidenced by changes in helix orientation; concanamycin additionally interacts with Vph1p TM7, suggesting that both stator and rotor elements contribute to the inhibitor site within the membrane. Comparison of the peptide behavior in lipid bilayers is made with membranous subunit c assemblies of the 16-kDa proteolipid from Nephrops norvegicus, which can substitute functionally for Vma3p in S. cerevisiae.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

The first putative transmembrane segment of subunit c" (Vma16p) of the yeast V-ATPase is not necessary for function

The yeast vacuolar ATPase (V-ATPase) contains three proteolipid subunits: c (Vma3p), c' (Vma11p), and c" (Vma16p). Each subunit contains a buried glutamate residue that is essential for function, and these subunits are not able to substitute for each other in supporting activity. Subunits c and c' each contain four putative transmembrane segments (TM1-4), whereas subunit c" is predicted to contain five. To determine whether TM1 of subunit c" serves an essential function, a deletion mutant of Vma16p was constructed lacking TM1 (Vma16p-Delta TM1). Although this construct does not complement the loss of Vma3p or Vma11p, it does complement the loss of full-length Vma16p. Vacuoles isolated from the strain expressing Vma16p-Delta TM1 showed V-ATPase activity and proton transport greater than 80% relative to wild type and displayed wild type levels of subunits A and a, suggesting normal assembly of the V-ATPase complex. These results suggest that TM1 of Vma16p is dispensable for both activity and assembly of the V-ATPase. To obtain information about the topology of Vma16p, labeling of single cysteine-containing mutants using the membrane-permeable reagent 3-(N-maleimidylpropionyl)biocytin (MPB) and the -impermeable reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) was tested. Both the Cys-less form of Vma16p and eight single cysteine-containing mutants retained greater than 80% of wild type levels of activity. Of the eight mutants tested, two (S5C and S178C) were labeled by MPB. MPB-labeling of S5C was blocked by AMS in intact vacuoles, whereas S178C was blocked by AMS only in the presence of permeabilizing concentrations of detergent. In addition, a hemagglutinin epitope tag introduced into the C terminus of Vma16p was recognized by an anti-hemagglutinin antibody in intact vacuolar membranes, suggesting a cytoplasmic orientation for the C terminus. These results suggest that subunit c" contains four rather than five transmembrane segments with both the N and C terminus on the cytoplasmic side of the membrane.     

Structural properties of a peptide derived from H-V-ATPase subunit a

The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H⁺-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The helical region extends well beyond A738, as was previously suggested based on NMR studies of a similar peptide in DMSO. The pKa of both histidine residues that are important for proton transport was measured in water and in SDS. The differences that are found demonstrate that the histidine residues interact with the SDS polar heads. In detergent, circular dichroism data indicate that the secondary structure of the peptide depends on the pH and the type of detergent used. Using solid-state NMR, it is shown that the peptide is immobile in phospholipid bilayers, which means that it is probably not a single transmembrane helix in these samples. The environment is important for the structure of TM7, so in subunit a it is probably held in place by the other transmembrane helices of this subunit.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase

Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Single replacement constructs of all hydroxyl, basic, and acidic amino acids identify new function and structure-sensitive regions of the mitochondrial phosphate transport protein

The phosphate transport protein (PTP) catalyzes the proton cotransport of phosphate into the mitochondrial matrix. It functions as a homodimer, and thus residues of the phosphate and proton pores are somewhat scattered throughout the primary sequence. With 71 new single mutation per subunit PTPs, all its hydroxyl, basic, and acidic residues have now been replaced to identify these essential residues. We assayed the initial rate of pH gradient-dependent unidirectional phosphate transport activity and the liposome incorporation efficiency (LIE) of these mutants. Single mutations of Thr79, Tyr83, Lys90, Tyr94, and Lys98 inactivate transport. The spacings between these residues imply that they are located along the same face of transmembrane (TM) helix B, requiring an extension of its current model C-terminal domain by 10 residues. This extension superimposes very well onto the shorter bovine PTP helix B, leaving a 15-residue hydrophobic extension of the yeast helix B N-terminus. This is similar to the helix D and F regions of the yeast PTP. Only one transport-inhibiting mutation is located within loops: Ser158Thr in the matrix loop between helices C and D. All other transport-inhibiting mutations are located within the TM helices. Mutations that yield LIEs of <6% are all, except for four, within helices. The four exceptions are Tyr12Ala near the PTP N-terminus and Arg159Ala, Glu163Gln, and Glu164Gln in the loop between helices C and D. The PTP C-terminal segment beyond Thr214 at the N-terminus of helix E has 11 mutations with LIEs >20% and none with LIE <6%. Mutations with LIEs >20% are located near the ends of all the TM helices except TM helix D. Only a few mutations alter PTP structure (LIE) and also affect PTP transport activity. A novel observation is that Ser4Ala blocks the formation of PTP bacterial inclusion bodies.     

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V₁ domain that hydrolyzes ATP and an integral V₀ domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V₀) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V₁V₀ complex and the free V₀ domain to identify residues protected from modification by the presence of V₁. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V₁ domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.     

Competition between neighboring topogenic signals during membrane protein insertion into the ER

To better define the mechanism of membrane protein insertion into the membrane of the endoplasmic reticulum, we measured the kinetics of translocation across microsomal membranes of the N-terminal lumenal tail and the lumenal domain following the second transmembrane segment (TM2) in the multispanning mouse protein Cig30. In the wild-type protein, the N-terminal tail translocates across the membrane before the downstream lumenal domain. Addition of positively charged residues to the N-terminal tail dramatically slows down its translocation and allows the downstream lumenal domain to translocate at the same time as or even before the N-tail. When TM2 is deleted, or when the loop between TM1 and TM2 is lengthened, addition of positively charged residues to the N-terminal tail causes TM1 to adopt an orientation with its N-terminal end in the cytoplasm. We suggest that the topology of the TM1-TM2 region of Cig30 depends on a competition between TM1 and TM2 such that the transmembrane segment that inserts first into the ER membrane determines the final topology.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

Structural contribution of C-terminal segments of NuoL (ND5) and NuoM (ND4) subunits of complex I from Escherichia coli

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.     

Segment TM7 from the cytoplasmic hemi-channel from VO-H+-V-ATPase includes a flexible region that has a potential role in proton translocation

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY (1)H-(1)H NMR spectra are obtained of sMTM7 dissolved in d(6)-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two alpha-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.     

Mechanics of coupling proton movements to c-ring rotation in ATP synthase

F1F0 ATP synthases generate ATP by a rotary catalytic mechanism in which H+ transport is coupled to rotation of an oligomeric ring of c subunits extending through the membrane. Protons bind to and then are released from the aspartyl-61 residue of subunit c at the center of the membrane. Subunit a of the F0 sector is thought to provide proton access channels to and from aspartyl-61. Here, we summarize new information on the structural organization of Escherichia coli subunit a and the mapping of aqueous-accessible residues in the second, fourth and fifth transmembrane helices (TMHs). Aqueous-accessible regions of these helices extend to both the cytoplasmic and periplasmic surface. We propose that aTMH4 rotates to alternately expose the periplasmic or cytoplasmic half-channels to aspartyl-61 of subunit c during the proton transport cycle. The concerted rotation of interacting helices in subunit a and subunit c is proposed to be the mechanical force driving rotation of the c-rotor, using a mechanism akin to meshed gears.     

Segment TM7 from the cytoplasmic hemi-channel from VO-H-V-ATPase includes a flexible region that has a potential role in proton translocation

A 900-MHz NMR study is reported of peptide sMTM7 that mimics the cytoplasmic proton hemi-channel domain of the seventh transmembrane segment (TM7) from subunit a of H⁺-V-ATPase from Saccharomyces cerevisiae. The peptide encompasses the amino acid residues known to actively participate in proton translocation. In addition, peptide sMTM7 contains the amino acid residues that upon mutation cause V-ATPase to become resistant against the inhibitor bafilomycin. 2D TOCSY and NOESY ¹H-¹H NMR spectra are obtained of sMTM7 dissolved in d₆-DMSO and are used to calculate the three-dimensional structure of the peptide. The NMR-based structures and corresponding dynamical features of peptide sMTM7 show that sMTM7 is composed of two α-helical regions. These regions are separated by a flexible hinge of two residues. The hinge acts as a ball-and-joint socket and both helical segments move independently with respect to one another. This movement in TM7 is suggested to cause the opening and closing of the cytoplasmic proton hemi-channel and enables proton translocation.     

Structural properties of ribosomal protein L11 from Escherichia coli

Protein L11 has been isolated from the large subunit of the E. coli ribosome under non-denaturing conditions and studied by proton magnetic resonance spectroscopy, limited proteolysis, and fluorescence and UV spectroscopy. The protein consists of two domains, a tightly-folded N-terminal part and a C-terminal half with an extended and loosely folded conformation. It is likely that the N-terminal domain is located on the surface of the subunit whereas the C-terminal part is buried within the ribosomal structure. The two tyrosines in the N-terminal region behave as solvent-exposed residues, in good agreement with iodination studies on L11 in situ. It appears probable that the central region of L11, in which the protease cleavages occur, plays an important part in structural and functional aspects.     

Arrangement of subunits in the proteolipid ring of the V-ATPase

The vacuolar ATPases (V-ATPases) are multisubunit complexes containing two domains. The V(1) domain (subunits A-H) is peripheral and carries out ATP hydrolysis. The V(0) domain (subunits a, c, c', c', d, and e) is membrane-integral and carries out proton transport. In yeast, there are three proteolipid subunits as follows: subunit c (Vma3p), subunit c' (Vma11p), and subunit c' (Vma16p). The proteolipid subunits form a six-membered ring containing single copies of subunits c' and c' and four copies of subunit c. To determine the possible arrangements of proteolipid subunits in V(0) that give rise to a functional V-ATPase complex, a series of gene fusions was constructed to constrain the arrangement of pairs of subunits in the ring. Fusions containing c' employed a truncated version of this protein lacking the first putative transmembrane helix (which we have shown previously to be functional), to ensure that the N and C termini of all subunits were located on the luminal side of the membrane. Fusion constructs were expressed in strains disrupted in c', c', or both but containing a wild copy of c to ensure the presence of the required number of copies of subunit c. The c-c'(DeltaTM1), c'(DeltaTM1)-c', and c'-c constructs all complemented the vma(-) phenotype and gave rise to complexes possessing greater than 25% of wild-type levels of activity. By contrast, neither the c-c', the c'-c'(DeltaTM1), nor the c'(DeltaTM1)-c constructs complemented the vma(-) phenotype. These results suggest that functionally assembled V-ATPase complexes contain the proteolipid subunits arranged in a unique order in the ring.     

The N-terminal region of the transmembrane domain of human erythrocyte band 3. Residues critical for membrane insertion and transport activity

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G(381)LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.