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Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Rahman S, Yamato I, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Murata T - PLoS ONE (2013)

Bottom Line: The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis.We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities.No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan. srahman@rs.tus.ac.jp

ABSTRACT
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.

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Structural model of human V-ATPase showing KD values for affinities of subunit-subunit interactions.Diverse affinities between subunits of the peripheral stalk of human V-ATPase are illustrated. Dotted arrows indicate weak and solid arrows strong binding interactions.
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pone-0055704-g007: Structural model of human V-ATPase showing KD values for affinities of subunit-subunit interactions.Diverse affinities between subunits of the peripheral stalk of human V-ATPase are illustrated. Dotted arrows indicate weak and solid arrows strong binding interactions.

Mentions: A quaternary complex of E1G1, C1, H, and a1NT was not sufficiently stable to be detected by gel filtration (Figure 6A–C); no peak was observed before the position of the other strong complexes (E1G1-C1 or E1G1-H). Almost all of the a1NT appeared at the tail position of the peak, indicating weak affinity of this subunit for the others (Figure 6B). Fractions analyzed by SDS-PAGE showed elution of H, C1, E1, and G1 at 1.85 ml (Figure 6C), with a1NT starting to emerge at approximately 1.95 ml (C1 and a1NT bands could be separated using a 7% gel, data not shown), slightly earlier than a1NT alone. The conditions used for native PAGE were very harsh to identify the weak binding interactions of a1NT with other subunits. The resultant strong band from the mixture of E1G1-C1-a1NT-H suggested that only E1G1, C1, and H were part of the complex, with the majority of unbound a1NT observed in its expected migratory position (Figure 6D). However, by pulldown assay, a quaternary complex eluted with the His-tagged H subunit was observed, although the band intensities of C1 and a1NT were relatively low. Bands of C1 and a1NT could be slightly separated, and the intensities of both of these bands were much stronger (Figure 6E, lane 2) than the previous investigation of ternary complex formation (Figure 4E and S7D). Taken together, we speculate that the sum of these individual interactions in the peripheral stalk region (Figure 7) is strong enough to withstand the torque of rotational catalysis and be able to maintain the structural integrity of the complex.


Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Rahman S, Yamato I, Saijo S, Mizutani K, Ishizuka-Katsura Y, Ohsawa N, Terada T, Shirouzu M, Yokoyama S, Iwata S, Murata T - PLoS ONE (2013)

Structural model of human V-ATPase showing KD values for affinities of subunit-subunit interactions.Diverse affinities between subunits of the peripheral stalk of human V-ATPase are illustrated. Dotted arrows indicate weak and solid arrows strong binding interactions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569449&req=5

pone-0055704-g007: Structural model of human V-ATPase showing KD values for affinities of subunit-subunit interactions.Diverse affinities between subunits of the peripheral stalk of human V-ATPase are illustrated. Dotted arrows indicate weak and solid arrows strong binding interactions.
Mentions: A quaternary complex of E1G1, C1, H, and a1NT was not sufficiently stable to be detected by gel filtration (Figure 6A–C); no peak was observed before the position of the other strong complexes (E1G1-C1 or E1G1-H). Almost all of the a1NT appeared at the tail position of the peak, indicating weak affinity of this subunit for the others (Figure 6B). Fractions analyzed by SDS-PAGE showed elution of H, C1, E1, and G1 at 1.85 ml (Figure 6C), with a1NT starting to emerge at approximately 1.95 ml (C1 and a1NT bands could be separated using a 7% gel, data not shown), slightly earlier than a1NT alone. The conditions used for native PAGE were very harsh to identify the weak binding interactions of a1NT with other subunits. The resultant strong band from the mixture of E1G1-C1-a1NT-H suggested that only E1G1, C1, and H were part of the complex, with the majority of unbound a1NT observed in its expected migratory position (Figure 6D). However, by pulldown assay, a quaternary complex eluted with the His-tagged H subunit was observed, although the band intensities of C1 and a1NT were relatively low. Bands of C1 and a1NT could be slightly separated, and the intensities of both of these bands were much stronger (Figure 6E, lane 2) than the previous investigation of ternary complex formation (Figure 4E and S7D). Taken together, we speculate that the sum of these individual interactions in the peripheral stalk region (Figure 7) is strong enough to withstand the torque of rotational catalysis and be able to maintain the structural integrity of the complex.

Bottom Line: The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis.We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities.No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan. srahman@rs.tus.ac.jp

ABSTRACT
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.

Show MeSH
Related in: MedlinePlus