Insertion of the plant photosystem I subunit G into the thylakoid membrane

Subunit G of photosystem I is a nuclear-encoded protein, predicted to form two transmembrane alpha-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosystem I subunit G (PSI-G). However, the presence of the tags in the loop make the membrane-inserted protein significantly more sensitive to trypsin, apparently by disrupting the interaction between the loop and the PSI core. Knock-out plants lacking PSI-G were transformed with constructs encoding the C-terminal and loop-tagged PSI-G proteins. Experiments on thylakoids from the transgenic lines show that the C-terminally tagged versions of PSI-G adopt the same topology as wild-type PSI-G, whereas the loop-tagged versions affect the sensitivity of the loop region to trypsin, thus confirming the in vitro observations. Furthermore, purification of PSI complexes from transgenic plants revealed that all the tagged versions of PSI-G are incorporated and retained in the PSI complex, although the C-terminally tagged variants of PSI-G were preferentially retained. This suggests that the loop region of PSI-G is important for proper integration into the PSI core. Our experiments demonstrate that it is possible to produce His- and Strep-tagged PSI in plants, and provide further evidence that the topology of membrane proteins is dictated by the distribution of positive charges, which resist translocation across membranes.     

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase.     

The stable assembly of newly synthesized PsaE into the photosystem I complex occurring via the exchange mechanism is facilitated by electrostatic interactions

Photosystem I (PSI) is a photochemically active membrane protein complex that functions at the reducing site of the photosynthetic electron-transfer chain as plastocyanin-ferredoxin oxidoreductase. PsaE, a peripheral subunit of the PSI complex, plays an important role in the function of PSI. PsaE is involved in the docking of ferredoxin/flavodoxin to the PSI complex and also participates in the cyclic electron transfer around PSI. The molecular characterization of the assembly of newly synthesized PsaE in the thylakoid membranes or in isolated PSI complexes is the subject of the present study. For this purpose the Mastigocladus laminosus psaE gene was cloned and overexpressed in Escherichia coli, and the resulting PsaE protein was purified to homogeneity by affinity chromatography. The purified PsaE was then introduced into thylakoids isolated from M. laminosus, and the newly introduced PsaE subunit saturates the membrane. The solubilization and separation of the different thylakoid protein complexes indicated that PsaE accumulates specifically in its functional location, the PSI complex. A similar stable assembly was detected when PsaE was introduced into purified PSI complexes, i.e., in the absence of other thylakoid components. This strongly indicates that the information for the stable assembly of PsaE into PSI lies within the polypeptide itself and within other subunits of the PSI complex that interact with it. To determine the nature of these interactions, the assembly reaction was performed in conditions affecting the ionic/osmotic strength. We found that altering the ionic strength significantly affects the capability of PsaE to assemble into isolated thylakoids or PSI complexes, strongly supporting the fact that electrostatic interactions are formed between PsaE and other PSI subunits. Moreover, the data suggest that the formation of electrostatic interactions occurs concomitantly with an exchange step in which newly introduced PsaE replaces the subunit present in situ.     

Photosystem I lacking the PSI-G subunit has a higher affinity for plastocyanin and is sensitive to photodamage

PSI-G is an 11 kDa subunit of PSI in photosynthetic eukaryotes. Arabidopsis thaliana plants devoid of PSI-G have a decreased PSI content and an increased activity of NADP(+) photoreduction in vitro but otherwise no obvious phenotype. To investigate the biochemical basis for the increased activity, the kinetic parameters of the reaction between PSI and plastocyanin were determined. PSI-G clearly plays a role in the affinity for plastocyanin since the dissociation constant (K(D)) is only 12 muM in the absence of PSI-G compared to 32 muM for the wild type. On the physiological level, plants devoid of PSI-G have a more reduced Q(A). This indicates that the decreased PSI content is due to unstable PSI rather than an adaptation to the increased activity. In agreement with this indication of decreased stability, plants devoid of PSI-G were found to be more photo-inhibited both at low temperature and after high light treatment. The decreased PSI stability was confirmed in vitro by measuring PSI activity after illumination of a thylakoid suspension which clearly showed a faster decrease in PSI activity in the thylakoids lacking PSI-G. Light response of the P700 redox state in vivo showed that in the absence of PSI-G, P700 is more reduced at low light intensities. We conclude that PSI-G is involved in the binding dynamics of plastocyanin to PSI and that PSI-G is important for the stability of the PSI complex.     

Photosystem I lacking the PSI-G subunit has a higher affinity for plastocyanin and is sensitive to photodamage

PSI-G is an 11 kDa subunit of PSI in photosynthetic eukaryotes. Arabidopsis thaliana plants devoid of PSI-G have a decreased PSI content and an increased activity of NADP⁺ photoreduction in vitro but otherwise no obvious phenotype [P.E. Jensen, L. Rosgaard, J. Knoetzel, H.V. Scheller, Photosystem I activity is increased in the absence of the PSI-G subunit. J. Biol. Chem. 277, (2002) 2798-2803.]. To investigate the biochemical basis for the increased activity, the kinetic parameters of the reaction between PSI and plastocyanin were determined. PSI-G clearly plays a role in the affinity for plastocyanin since the dissociation constant (K_D) is only 12 μM in the absence of PSI-G compared to 32 μM for the wild type. On the physiological level, plants devoid of PSI-G have a more reduced Q_A. This indicates that the decreased PSI content is due to unstable PSI rather than an adaptation to the increased activity. In agreement with this indication of decreased stability, plants devoid of PSI-G were found to be more photoinhibited both at low temperature and after high light treatment. The decreased PSI stability was confirmed in vitro by measuring PSI activity after illumination of a thylakoid suspension which clearly showed a faster decrease in PSI activity in the thylakoids lacking PSI-G. Light response of the P700 redox state in vivo showed that in the absence of PSI-G, P700 is more reduced at low light intensities. We conclude that PSI-G is involved in the binding dynamics of plastocyanin to PSI and that PSI-G is important for the stability of the PSI complex.     

An insight into the assembly and organization of photosystem I complex in the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus

The present study characterizes the assembly and organization of Photosystem I (PSI) complex, and its individual subunits into the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus. PSI is a multiprotein complex that contains peripheral as well as integral subunits. Hence, it serves as a suitable model system for understanding the formation and organization of membrane protein complexes. In the present study, two peripheral cytosol facing subunits of PSI, namely, PsaD and PsaE were overexpressed in E. coli and used for assembly studies. The gene encoding PsaK, an integral membrane spanning subunit of PSI, was cloned and the deduced amino acid sequence revealed PsaK to have two transmembrane alpha-helices. The characterization of the in vitro assembly of the peripheral subunits, PsaD and PsaE, as well as of the integral subunit, PsaK, was performed by incubating each subunit with thylakoids isolated from Mastigocladus laminosus. All three subunits studied were found to assemble into the thylakoids in a spontaneous mechanism, showing no requirement for cytosolic factors or NTP's (nucleotide 5'-triphosphate). Nevertheless, further characterization of the assembly of PsaK revealed its membrane integration to be most efficient at 55 degrees C. The associations and protein-protein interactions between different subunits within the assembled PSI complex were directly quantified by measurements performed using the BIACORE technology. The preliminary results indicated the existence of specific interaction between PsaD and PsaE, and revealed a very high binding affinity between PsaD and the PSI electron acceptor ferridoxin (Kd = 5.8 x 10(-11) M). PsaE has exhibited a much lower binding affinity for ferridoxin (Kd = 3.1 x 10(-5) M), thereby supporting the possibility of PsaE being one of the subunits responsible for the dissociation of ferridoxin from the PSI complex.     

Single and double knockouts of the genes for photosystem I subunits G,K, and H of Arabidopsis. Effects on photosystem I composition,photosynthetic electron flow,and state transitions

Photosystem I (PSI) of higher plants contains 18 subunits. Using Arabidopsis En insertion lines, we have isolated knockout alleles of the genes psaG, psaH2, and psaK, which code for PSI-G, -H, and -K. In the mutants psak-1 and psag-1.4, complete loss of PSI-K and -G, respectively, was confirmed, whereas the residual H level in psah2-1.4 is due to a second gene encoding PSI-H, psaH1. Double mutants, lacking PSI-G, and also -K, or a fraction of -H, together with the three single mutants were characterized for their growth phenotypes and PSI polypeptide composition. In general, the loss of each subunit has secondary, in some cases additive, effects on the abundance of other PSI polypeptides, such as D, E, H, L, N, and the light-harvesting complex I proteins Lhca2 and 3. In the G-less mutant psag-1.4, the variation in PSI composition suggests that PSI-G stabilizes the PSI-core. Levels of light-harvesting complex I proteins in plants, which lack simultaneously PSI-G and -K, indicate that PSI subunits other than G and K can also bind Lhca2 and 3. In the same single and double mutants, psag-1.4, psak-1, psah2-1.4, psag-1.4/psah2-1.4, and psag-1.4/psak-1 photosynthetic electron flow and excitation energy quenching were analyzed to address the roles of the various subunits in P700 reduction (mediated by PSI-F and -N) and oxidation (PSI-E), and state transitions (PSI-H). Based on the results, we also suggest for PSI-K a role in state transitions.     

Insertion of PsaK into the thylakoid membrane in a "Horseshoe" conformation occurs in the absence of signal recognition particle, nucleoside triphosphates, or functional albino3.

The photosystem I subunit PsaK spans the thylakoid membrane twice, with the N and C termini both located in the lumen. The insertion mechanism of a thylakoid membrane protein adopting this type of topology has not been studied before, and we have used in vitro assays to determine the requirements for PsaK insertion into thylakoids. PsaK inserts with high efficiency and we show that one transmembrane span (the C-terminal region) can insert independently of the other, indicating that a "hairpin"-type mechanism is not essential. Insertion of PsaK does not require stromal extract, indicating that signal recognition particle (SRP) is not involved. Removal of nucleoside triphosphates inhibits insertion only slightly, both in the presence and absence of stroma, suggesting a mild stimulatory effect of a factor in the translation system and again ruling out an involvement of SRP or its partner protein, FtsY. We, furthermore, find no evidence for the involvement of known membrane-bound translocation apparatus; proteolysis of thylakoids destroys the Sec and Tat translocons but does not block PsaK insertion, and antibodies against the Oxa1/YidC homolog, Alb3, block the SRP-dependent insertion of Lhcb1 but again have no effect on PsaK insertion. Because YidC is required for the efficient insertion of every membrane protein tested in Escherichia coli (whether SRP-dependent or -independent), PsaK is the first protein identified as being independent of YidC/Alb3-type factors in either thylakoids or bacteria. The data raise the possibility of a wholly spontaneous insertion pathway.     

The assembly of the PsaD subunit into the membranal photosystem I complex occurs via an exchange mechanism.

PsaD is a peripheral stromal-facing subunit of photosystem I (PSI), a multisubunit complex of the thylakoid membranes. PsaD plays a major role in both the function and assembly of PSI. Past studies with radiolabeled PsaD indicated that PsaD is able to assemble in vitro specifically into the PSI complex. To unravel the mechanism by which this assembly takes place, the following steps were taken. (i) Mature PsaD of spinach and PsaD of the prokaryotic caynobacterium Mastigocladus laminosus, both bearing a six-histidine tag at their C-termini, were overexpressed in Escherichia coli and purified to homogeneity. (ii) The purified recombinant protein was introduced into the isolated PSI complex. (iii) Following incubation, the PsaD that assembled into PSI was separated from the nonassembled PsaD by a sucrose gradient. Differential Western blot analysis was used to determine whether the native and the recombinant PsaD were present as free or assembled proteins of the PSI complex. Antibodies that can recognize only the recombinant PsaD (anti-his) or both the native and recombinant PsaD (anti-PsaD) were used. The findings indicated that an exchange mechanism enables the assembly of a newly introduced PsaD into PSI. The latter replaces the PsaD subunit that is present in situ within the complex. In vivo studies that followed the assembly of PsaD in Chlamydomonas reinhardtii cells supported this in vitro-characterized exchange mechanism. In C. reinhardtii, in the absence of synthesis and assembly of new PSI complexes, newly synthesized PsaD assembled into pre-existing PSI complexes.     

Assembly and processing of subunit II (PsaD) precursor in the isolated photosystem-I complex.

The precursor of photosystem I (PSI) subunit II (pre-subunit II) synthesized in vitro, was found to bind to the holo-PSI complex, both within the thylakoids and outside, after detergent extraction of PSI from the membranes. Chloroplast stromal fraction added to the purified PSI complexes, containing the labeled pre-subunit II, induced the processing of the precursor to the mature form. This implies that processing can occur within the isolated complex, after the integration of the precursor. The results presented suggest that certain aspects of biogenesis of membranal protein complexes can be studied in detergent-extracted purified complexes.     

Charge Composition Features of Model Single-span Membrane Proteins That Determine Selection of YidC and SecYEG Translocase Pathways in Escherichia coli

We have investigated the features of single-span model membrane proteins based upon leader peptidase that determines whether the proteins insert by a YidC/Sec-independent, YidC-only, or YidC/Sec mechanism. We find that a protein with a highly hydrophobic transmembrane segment that inserts into the membrane by a YidC/Sec-independent mechanism becomes YidC-dependent if negatively charged residues are inserted into the translocated periplasmic domain or if the hydrophobicity of the transmembrane segment is reduced by substituting polar residues for nonpolar ones. This suggests that charged residues in the translocated domain and the hydrophobicity within the transmembrane segment are important determinants of the insertion pathway. Strikingly, the addition of a positively charged residue to either the translocated region or the transmembrane region can switch the insertion requirements such that insertion requires both YidC and SecYEG. To test conclusions from the model protein studies, we confirmed that a positively charged residue is a SecYEG determinant for the endogenous proteins ATP synthase subunits a and b and the TatC subunit of the Tat translocase. These findings provide deeper insights into how pathways are selected for the insertion of proteins into the Escherichia coli inner membrane.     

Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions.     

Unique structural determinants in the signal peptides of "spontaneously" inserting thylakoid membrane proteins.

A series of thylakoid membrane proteins, including PsbX, PsbY and PsbW, are synthesized with cleavable signal peptides yet inserted using none of the known Sec/SRP/Tat/Oxa1-type insertion machineries. Here, we show that, although superficially similar to Sec-type signal peptides, these thylakoidal signal peptides contain very different determinants. First, we show that basic residues in the N-terminal domain are not important, ruling out electrostatic interactions as an essential element of the insertion mechanism, and implying a fundamentally different targeting mechanism when compared with the structurally similar M13 procoat. Second, we show that acidic residues in the C-domain are essential for the efficient maturation of the PsbX and PsbY-A1 peptides, and that even a single substitution of the -5 Glu by Val in the PsbX signal peptide abolishes maturation in the thylakoid. Processing efficiency is restored to an extent, but not completely, by the highly hydrophilic Asn, implying that this domain is required to be hydrophilic, but preferably negatively charged, in order to present the cleavage site in an optimal manner. We show that substitution of the PsbX C-domain Glu residues by Val leads to a burial of the cleavage site within the bilayer although insertion is unaffected. Finally, we show that substitution of the Glu residues in the lumenal A2 loop of the PsbY polyprotein leads to a block in cleavage on the stromal side of the membrane, and present evidence that the PsbY-A2 signal peptide is required to be relatively hydrophilic and unable to adopt a transmembrane conformation on its own. These data indicate that, rather than being merely additional hydrophobic regions to promote insertion, the signal peptides of these thylakoid proteins are complex domains with uniquely stringent requirements in the C-domain and/or translocated loop regions.     

Tim23p Contains Separate and Distinct Signals for Targeting to Mitochondria and Insertion into the Inner Membrane

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.     

Topography of the protein complexes of the chloroplast thylakoid membrane : studies of photosystem I using a chemical probe and proteolytic digestion

The transverse heterogeneity of the polypeptides associated with the Photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation has been studied using the impermeant chemical modifier, 2,4,6-trinitrobenzenesulfonate (TNBS) and the proteolytic enzyme, Pronase E. The present study has shown that the PSI reaction center polypeptide of ~62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light-harvesting chlorophyll protein (LHCPI) complex are not labeled by [¹⁴C]TNBS in unfractionated thylakoids. On the other hand, the 23 kilodalton polypeptide of the PSI LHCP and the 19 and 14 kilodalton polypeptides associated with the PSI primary electron acceptor complex are readily labeled by [¹⁴C]TNBS and are exposed to the stromal side of the thylakoid. Differences and similarities in the labeling of polypeptides associated with the PSI complex in thylakoids and in the isolated PSI complex are also noted. Treatment of thylakoids with pronase had no effect on the organization of the polypeptides in the LHCPI or the reaction center core complex, as manifested by the separation of these two subcomplexes from pronase-treated membranes. The 62, 19, and 14 kilodalton polypeptides associated with the reaction center core complex and the 23 and 22 kilodalton polypeptides associated with LHCPI are sensitive to pronase treatment while the 20 kilodalton polypeptide of LHCPI was inaccessible to the protease. The proteolysis of the 62 kilodalton polypeptide generated first a single immunodetectable fragment at about 48 kilodaltons, and further proteolytic digestion generated two other fragments at 30 and 17 kilodaltons respectively. These results are discussed in relation to the organization of the PSI complex in spinach thylakoids. A model for the transmembrane topography of the polypeptide constituents of PSI has been developed.     

A negatively charged N terminus in the alpha polypeptide inhibits formation of light-harvesting complex I in Rhodobacter capsulatus.

Light-harvesting complex I (LHI) of Rhodobacter capsulatus contains bacteriochlorophyll and carotenoids which are noncovalently bound to two different apoproteins (alpha and beta polypeptides) carrying oppositely charged N-terminal ends. The contribution of these charged segments to the assembly of LHI was studied with mutants having oppositely charged amino acids in the alpha or beta polypeptide. The influence of these mutations on the insertion and assembly process of the LHI complex was investigated by means of spectroscopic analysis of isolated intracytoplasmic membranes and pulse-chase experiments. Exchange of four positively charged amino acids to negatively charged amino acids on the N-terminal domain of the alpha subunit inhibited completely the assembly of the LHI complex. Although this mutant has no antenna, the reaction center is active and the cells were able to grow anaerobically in the light. Conversely, mutation of the four negatively charged amino acids of the N-terminal segment of the beta polypeptide did not prevent the assembly of the LHI complex, although the stability of the complex and the size of the photosynthetic unit were affected. The presence of the mutated beta polypeptide was confirmed by protein sequencing.     

Insertion of the precursor of the light-harvesting chlorophylla/b-protein into the thylakoids requires the presence of a developmentally regulated stromal factor

The precursor of the major light-harvesting chlorophylla/b-proteins of photosystem II was synthesizedin vitro from a gene fromLemna gibba. When the labelled precursor was incubated with developing barley plastids, the precursor and the processed polypeptide were incorporated in the thylakoids in proportions that varied depending on the developmental stage of plastids. At early stages of development most of the precursor associated with the thylakoids could be removed by washing with 0.1 M NaOH, while in more mature plastids most of its was resistant to a NaOH wash. Insertion of the precursor into thylakoids required the presence of a stromal factor and Mg-ATP. The stromal factor is probably a protein. The insertion reaction has an optimal temperature of 25°C and a pH of 8. The appearance of the stromal factor and the thylakoid membrane's receptivity for the insertion of the precursor depended on the stage of plastid development. These observations are consistent with the hypothesis that the insertion of the precursor into the thylakoid prior to its proteolytic processing, is one of the steps involved in the assembly of the light-harvesting complex of photosystem II.     

Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34. The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex.