Defined subcomplexes of the A<sub>1</sub> ATPase from the archaeon <i>Methanosarcina mazei</i> Gö1: biochemical properties and redox regulation

117

Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92-959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca 2؉ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.After the proposed endosymbiotic event that gave rise to chloroplasts and, as a consequence, to the massive transfer of genes from the endosymbiont to the host cell nucleus, two new machineries were developed at the outer and inner envelope of chloroplasts, the TOC and TIC complexes, respectively. The machineries transport plastid proteins, whose synthesis was moved to the cytosol, back to the plastid (for a review, see Refs. 1 and 2). Whereas the channel of the TOC complex is homologous to the Omp85 family of transporters in bacteria (3), the TIC complex shows no homology to known transport systems, since the plasma membrane-bacterial transport machineries were relocated to the thylakoid membrane. Moreover, there was an essential requirement for establishing new lines of communication between the organelle and other parts of the cell (4). In chloroplasts of land plants, redox regulation is linked to oxygen and/or light. The regulation of chloroplast-specific processes by redox in accord with the status of the organelle applies not only for transcription and translation but also for translocation. Redox regulation at the translocation level has been proposed for the TIC machinery on the basis of protein composition and dynamics (5-8) as well as specific in vitro import experiments (9). However, despite advances in our biochemical understanding of the import process, little is presently known regarding details of the role of redox or of the structure of the TIC complex, particularly with respect to its channel subunits.Tic110 is essential for protein import into plastids (10 -12). It has been proposed to be a pore-forming protein and one of the main components of the TIC complex (13). Previous studies have provided information about certain aspects of Tic110 structure-function. The region from residu...

Section: Native Tic110 In Iev Is Sensitive To Proteases That Cannot Csupporting

confidence: 83%

Characterization of Tic110, a Channel-forming Protein at the Inner Envelope Membrane of Chloroplasts, Unveils a Response to Ca2+ and a Stromal Regulatory Disulfide Bridge

Balsera

Goetze

Kovács-Bogdán

et al. 2009

117

“…The fragment containing genes ahaA, ahaB, ahaF, and ahaD of MmA 3 B 3 DF-M1 were synthesized and provided by DNA 2.0 in the vector pJ367 (in-house cloning vector). The fragment was further subcloned into pGEM-4Z (35). The pGEM-4Z vector was kindly provided by Prof. V. Müller (Johann-Wolfgang Goethe University, Frankfurt, Germany).…”

Section: Methodsmentioning

confidence: 99%

Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors

Sielaff

Martin

Singh

et al. 2016

Self Cite

Edited by Velia FowlerThe angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A 3 B 3 DF, from the Methanosarcina mazei Gö1 A-ATP synthase, and the thermophilic motor ␣ 3 ␤ 3 ␥, from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase, are resolved at 5 s resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and they differ only in the magnitude of their velocities. M. mazei A 3 B 3 DF power strokes occurred in 120°steps at saturating ATP concentrations like the F-type motors. However, because ATP-binding dwells did not interrupt the 120°steps at limiting ATP, ATP binding to A 3 B 3 DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40°after the catalytic dwell. In F-type motors, elevated ADP induces dwells 40°after the catalytic dwell and slows the overall velocity. The similarities in these power stroke profiles are consistent with a common rotational mechanism for A-type and F-type rotary motors, in which the angular velocity is limited by the rotary position at which ATP binding occurs and by the drag imposed on the axle as it rotates within the ring of stator subunits.The primary source of ATP in archaea such as methanogens is the A 1 A O -ATP synthase, which shares several structural features with eukaryotic V 1 V O -ATPases and is evolutionarily distant from the F 1 F O -ATP synthases that fulfill this role in other bacteria and eukaryotes (1, 2). Metabolism in archaea is coupled to the generation of H ϩ and/or Na ϩ potentials across the membrane, both of which can provide the energy for ATP synthesis by the A 1 A O -ATP synthase. Whereas the F 1 F O -ATP synthases in prokaryotes and eukaryotes catalyze ATP synthesis at the expense of an electrochemical ion potential, the evolutionarily related V 1 V O -ATPases function as ATP-driven ion pumps and are unable to synthesize ATP under physiological conditions (3)(4)(5). Although the cellular function of archaeal ATP synthases is to synthesize ATP powered by a non-equilibrium electrochemical gradient, they also work as ATP-driven ion pumps to generate an ion gradient under fermentative conditions (6).The A-ATP synthases are composed of two opposed rotary motors as follows: an integral membrane A O complex (ac x ) involved in ion translocation, and a peripheral A 1 complex (A 3 B 3 CDF) that contains the catalytic sites for a total of nine different subunits (6). Both motors are connected by two peripheral stalks each composed of an EH heterodimer. These complexes share common features with bacterial

“…The molar ratio of the five subunits is A 3 :B 3 :C:D:F (Fig. 1B), based on a quantitation of the staining intensity of the five bands of these subunits, indicating the improvement of this new isolation protocol, when compared with a previous preparation in which subunit C is present in substoichiometric amounts (6). The contaminating bands running above subunit A are identified as E. coli DnaK and Grp E chaperons (data not shown).…”

Section: Isolation Andmentioning

confidence: 88%

“…Characterization of the A 1 -ATPase-The A 1 -ATPase has been purified by modification of an earlier method (6). The main differences from the earlier procedure are that the gel filtration column BioPrepSE1000/17 and the anionexchange column were replaced.…”

Section: Isolation Andmentioning

confidence: 99%

“…This class of enzymes is different from F 1 F 0 -or V 1 V 0 -ATPases by function, subunit composition, regulation, and structure (1). The A 1 A 0 -ATPase has at least nine subunits (A 3 :B 3 :C:D:E:F:H:I:K x ), but the actual subunit stoichiometry, especially regarding the proteolipid subunits K in AATPases, is different in various organisms (12,6,4 or, as suggested by genomic data, only 1 (5)). As suggested by its bipartite name, the A 1 A 0 -ATPase is composed of a water-soluble A 1 -ATPase and an integral membrane subcomplex, A 0 .…”

mentioning

confidence: 99%

See 1 more Smart Citation

Three-dimensional Organization of the Archaeal A1-ATPase from Methanosarcina mazei Gö1

Coskun

Radermacher

Müller

et al. 2004

Self Cite

Methanogenic, halophilic, and thermophilic Archaea synthesize ATP by means of ion gradient-driven phosphorylation. Although it was speculated for some time, due to the lack of indepth information, that the ATP synthases of Archaea may be either F 1 F 0 -or V 1 V 0 -like enzymes, it is now clear that they evolved as a separate class of ATPases/ATP synthases, the A 1 A 0 -ATPase/synthases (1-4). This class of enzymes is different from F 1 F 0 -or V 1 V 0 -ATPases by function, subunit composition, regulation, and structure (1). The A 1 A 0 -ATPase has at least nine subunits (A 3 :B 3 :C:D:E:F:H:I:K x ), but the actual subunit stoichiometry, especially regarding the proteolipid subunits K in AATPases, is different in various organisms (12, 6, 4 or, as suggested by genomic data, only 1 (5)). As suggested by its bipartite name, the A 1 A 0 -ATPase is composed of a water-soluble A 1 -ATPase and an integral membrane subcomplex, A 0 . ATP is synthesized or hydrolyzed on the A 1 headpiece, consisting of an A 3 B 3 domain, and the energy provided for or released during that process is transmitted to the membrane-bound A 0 domain (1). The energy coupling between the two active domains occurs via the so-called stalk part, an assembly proposed to be composed by the subunits C, D, and F (2). The archaeal A 1 A 0 -ATPase/synthase is regarded as a chimeric protein in which the membrane domain is closely related to F 1 F 0 -ATP synthases but the catalytic subunits closely to V 1 V 0 -ATPases (3, 4).The A 1 -ATPase from Methanosarcina mazei Gö1 is made up of at least the five different subunits A-D and F with apparent molecular masses of 65, 54, 41, 28, and 9 kDa (6). The enzyme, as shown by small angle x-ray scattering data, consists of an ϳ10-nm long headpiece and an 8.5-nm high and 6.0-nm diameter stalk (7). A comparison of the central stalk of this A 1 complex with bacterial F 1 -and V 1 -ATPase indicates different lengths of the stalk domain (7-9). The prevailing view is, however, that ATP synthesis/hydrolysis in the A 1 headpiece is coupled to ion flow in A 0 through rotational movements of the central stalk subunit(s) as demonstrated for the F 1 (reviewed in Refs. 10 and 11) and the Thermus thermophilus A 1 /V 1 -ATPase (12). (Note, the so-called V 1 V 0 -ATPase from T. thermophilus, which also synthesizes ATP, is of archaeal origin (13, 14); therefore, the ATPase headpiece will be considered A 1 /V 1 -ATPase throughout this work.)Here we describe a modified isolation procedure of the A 1 -ATPase from M. mazei Gö1, which facilitates the first threedimensional reconstruction of this enzyme by using tilt pairs of negatively stained molecules. The structure adds to the emerging picture of A 1 in which three copies of A and B subunits are arranged as a hexagonal barrel, enclosing a large cavity in which a shaft is asymmetrically located. This three-dimensional model allows comparison with structural models of related F 1 -and V 1 -ATPase determined by crystallography (15) and electron microscopy (16,17). Furthermore, insights i...