PhotosynthesisResearch 47:1-11, 1996. (~) 1996KluwerAcademicPublishers. Printedin the Netherlands. Minireview

The mechanism of Rubisco activase: Insights from studies of the properties and structure of the enzyme* M i c h a e l E. S a l v u c c i I & W i l l i a m L. O g r e n 2 1United States Department of Agriculture-Agricultural Research Service, Western Cotton Research Laboratory, 4135 E. Broadway Road, Phoenix, AZ 85040-8830; 2photosynthesis Research Unit, Urbana, IL 61801-3838, USA Received 12 September1995;acceptedin revisedform27 October1995

Key words: chaperone chloroplast, enzyme regulation, photosynthesis, protein-protein interaction, ribulose-1,5bisphosphate carboxylase/oxygenase

Abstract

Rubisco, the primary carboxylating enzyme in photosynthesis, must be activated to catalyze CO2 fixation. The concept of an 'activase', a specific protein for activating Rubisco, was first introduced in 1985 based largely on biochemical and genetic studies of a high CO2-requiring mutant ofArabidopsis (Salvucci et al. (1985) Photosynth Res 7: 193-201). Over the past ten years, details about the occurrence, structure, and properties of Rubisco activase have been elucidated. However, the mechanism of action of Rubisco activase remains elusive. This review discusses the need for and function of Rubisco activase and summarizes information about the properties and structure of Rubisco activase. The information is evaluated in the context of the mechanism of Rubisco activase.

Abbreviations: CA l - P - carboxyarabinitol 1-phosphate; PS - photosystem; Rubisco- ribulose 1,5-bisphosphate carboxylase/oxygenase; R u B P - ribulose 1,5-bisphosphate; X u B P - xylulose 1,5-bisphosphate Properties of Rubisco and the need for an activase

Brief historical perspective on Rubisco activation 1 The occurrence of Rubisco activation was recognized shortly after the enzyme was discovered, when Pon et al. (1963) observed a stimulation of activity following preincubation with HCO3 and Mg 2+. Later, Bahr and Jensen (1974) reported a 'low KM(CO2)' form when the enzyme was assayed in lysed chloroplasts. This observation was particularly important since a puzzling question at the time was how Rubisco catalyzed photosynthetic CO2 fixation at a high rate when the measured KM(CO2) was an order of magnitude greater than the atmospheric CO2 concentration. This dilemma was explained in part by the finding that Rubisco * The US Governmentright to retain a non-exclusive,royaltyfreelicencein and to any copyrightis acknowledged.

required activation by a CO2 molecule (Lorimer et al. 1976; Lorimer and Miziorko 1980) distinct from substrate CO2 (Lorimer 1979). Thus high KM(CO2) values were observed in early experiments because increasing CO2 concentration during assay not only provided more substrate but also activated more Rubisco.

Mechanistic considerations: Rubisco carbamylation and sugar-phosphate binding We now know that Rubisco must be activated by CO2 in order for it to catalyze the carboxylation or oxygenation of RuBP. The biochemical basis of this activation is carbamylation of an active site lysine residue (Lorimer and Miziorko 1980; Lorimer 1981). As shown in Fig. 1, the formation of a carbamate on the side-chain of a specific lysyl residue produces an anionic species capable of binding a divalent metal cation. The metal ion is essential for catalytic activity, but can only bind to Rubisco once the active site is carbamylated (reviewed

INACTIVE

ACTIVATED lmt

Rubisco I Lys-NH$+

Rubis¢o I Lys-Nll2

÷ CO 2

-3.

Rubis¢o ! Lys-Nil-O~O-

.~

Rubis¢o ! Lys-NH-ODO-Mg2+

÷ M~ +

(after Lorimer et a[. 1976)

Fig. 1. Biochemicalbasis for activationof Rubisco. The e-amineof lysine 201 in the active site of Rubisco is carbamylatedby reaction with CO2, creating an anionic species capable of binding the essential metal ion. Carbamylationfollowed by addition of the metal ion converts Rubisco from an inactiveto an activated form. in Andrews and Lorimer 1987; Hartman and Harpel 1994). Thus, carbamylation is a prerequisite for Rubisco activity via its required role in metal binding. In the absence of RuBP, carbamate formation occurs spontaneously upon incubation of isolated Rubisco with CO2 and Mg 2+. The concentration of CO2 required for half-maximal activation of isolated Rubisco (i.e. the Kact) is 23-40 #M at physiological H + and Mg 2+ concentrations (Hatch and Jensen 1980, Portis et al. 1986). The Rubisco active site exhibits a high degree of structural integrity even when the enzyme is not carbamylated (Schneider et al. 1992; Schreuder et al. 1993). Consequently, Rubisco does not require carbamylation to bind sugar-phosphates. In fact, some sugar-phosphates bind tighter to Rubisco when the active site is not carbamylated (reviewed in Portis 1992). Physiologically the most significant of these sugar-phosphates is the substrate, RuBP. The fight binding of RuBP to decarbamylated Rubisco prevents spontaneous carbamylation (Jordan and Chollet 1983) and tends to shift the overall equilibrium to the decarbamylated state (Brooks and Portis 1988; Portis 1992). The net result is that RuBP tends to stabilize Rubisco in an inactive form. Certain sugar-phosphates bind extremely tightly to Rubisco once the active site is carbamylated. These compounds are tight-binding competitive inhibitors that inhibit catalysis by preventing the substrate RuBP from binding to carbamylated enzyme. Sugarphosphates in this class include carboxyarabinitol 1phosphate (CA1-P) and 3-ketoarabinitol bisphosphate (Gutteridge et al. 1986; Berry et al. 1987; Edmondson et al. 1990a, b). Carboxyarabinitol 1-phosphate is a branched-chain sugar-phosphate that accumulates to relatively high levels in the leaves of some plant species (Moore et al. 1991). 3-ketoarabinitol bisphosphate is one of two so called 'fall-over '2 compounds

that form from misprotonation of RuBP at the active site of Rubisco (Edmondson et al. 1990a, b; Zhu and Jensen 1991a, b). The other 'fall-over' compound is xylulose bisphosphate, an isomer of RuBP. Xylulose bisphosphate is also a potent inhibitor of Rubisco by virtue of its fight binding to decarbamylated Rubisco at pH < 7.5 or carbamylated Rubisco at pH > 8.0 (Yokota 1991; Zhu and Jensen 1991a, b). Rubisco activase is required to facilitate activation of Rubisco in vivo Given its properties and the conditions that occur in the chloroplast, it is difficult to envision how Rubisco can function in vivo if activation occurred spontaneously. First, the level of CO2 in the chloroplast is considerably lower than the Kact(CO2), sufficient for only partial carbamylation of the enzyme. Second, even with adequate CO2 the high concentration of RuBP in the chloroplasts of illuminated leaves (Perchorowicz et al. 1981; Sicher et al. 1981) would make carbamylation very slow and difficult to maintain. Finally, chloroplasts contain CA1-P, XuBP and possibly other tightbinding inhibitors that block RuBP from binding to the enzyme once the active site is carbamylated. Together these conditions should reduce the catalytic activity of Rubisco to a very low level (Fig. 2). Instead, the observed rates of CO2 fixation at saturating irradiance indicate that Rubisco in the leaf routinely operates at 70-100% of maximal activity. Research over the past 10 years has shown that these high rates are possible because of the action of Rubisco activase, a nuclearencoded chloroplast protein that enables Rubisco to function under physiological conditions. Rubisco activase was first identified as the biochemical lesion responsible for the high CO2requiring phenotype of the rca mutant of Arabidopsis (Somerville et al. 1982), a mutant that lacks the Rubis-

activation level

activation [ level

100 .....

SO0

.

0 ""-"

.

.

.

.

-

0

no activase

activase + RuBP

(in vitro)

(in vivo)

Fig. 2. Factors affectingthe activationlevel in vitro and in vivo. In the absenceof RuBP, the activationlevel in vitro is dependenton the CO2 concentration. However,the decarbamylatedform of Rubisco is stabilizedand promotedby tight binding of RuBP leading to a low activation level. In vivo,the presenceof Rubiscoactivasepromotescarbamylationof Rnbiscoat low CO2 and in the presenceof RuBP. With activase,the actual level of Rubisco activationdependson the ATP/ADPratio.

co activase protein (Salvucci et al. 1985). Study of Rubisco in the mutant (Somerville et al. 1982; Salvucci et al. 1986) and in transgenic tobacco plants with reduced Rubisco activase levels (Mate et al. 1993) showed that Rubisco can not achieve and maintain an adequate level of activity for growth at ambient CO2 without the aid of Rubisco activase. In other words, Rubisco is largely inactive in vivo without an activase. This condition is consistent with predictions based on the in vitro properties of Rubisco and our understanding of the constitution of the chloroplast milieu. Rubisco activase promotes 'activation' of Rubisco by overcoming the deleterious effects of tightbinding sugar-phosphates and low chloroplast CO2 levels on catalysis and carbamylation (Fig. 2). Through a dependence on the levels of ATP and ADP, Rubisco modulates the extent of Rubisco activation in response to irradiance and other environmental factors (Machler and NSsberger 1980; Perchorowicz et al. 1981; Perchorowicz and Jensen 1983; Sharkey et al. 1986; Kobza and Edwards 1987; Sage et al. 1989; Schmieden-Kompalla et al. 1989; Parry et al. 1993). Regulation of Rubisco in this manner constitutes an important level of control on photosynthesis by ensuring that the rate of CO2 fixation is coordinately regulated with the rate of electron transport activity (Salvucci et al. 1985, 1987a; Robinson and Portis 1988). Coordinate regulation of these reactions improves the responsiveness of photosynthesis by maintaining metabolites at optimal levels through changes in environmen-

tal conditions (Perchorowicz et al. 1981; Salvucci et al. 1986; Sharkey et al. 1986; von Caemmerer and Edmondson 1986)

Structure and occurrence of Rubisco activase Structure

Rubisco activase is synthesized on cytosolic ribosomes and imported into the chloroplast. The polypeptide(s) is initially synthesized as a precursor and processed into a mature polypeptide of about 41 to 46 kDa by removal of the transit peptide (Werneke et al. 1988b). In many plant species, the mature Rubisco activase protein consists of two related polypeptides that arise from alternative pre-mRNA splicing at the intron nearest the 3 ~end of the RNA (Werneke et al. 1989). Thus, the longer polypeptide differs from the shorter by having an additional 35 or so amino acid residues at the carboxy terminus. The rca mutation is caused by a mutation at the 5~-splice junction of intron 3 which causes inefficient and incomplete splicing of the premRNA (Werneke et al. 1989; Orozco et al. 1993). Both isoforms of the spinach Rubisco activase gene have been cloned and expressed separately in E. coll. The individual cDNAs yield highly active enzyme, but the specific activity of the 41-kDa isoform for Rubisco activation is about 1.5- to 3-fold greater than the 45kDa polypeptide (Shen et al. 1991). The basis of the

altered interaction between Rubisco and the two forms of Rubisco activase clearly resides in the carboxy terminus, but the significance of the two isoforms is not known. Western immunoblots of extracts from a few species, such as tobacco and Chlamydomonas, indicate the presence of only a single 41- or 42-kDa polypeptide (Salvucci et al. 1987b; Roesler and Ogren 1990). These observations suggest that the larger polypeptide is not essential, yet the unorthodox mechanism of synthesis of the two forms by alternative splicing remains highly conserved. Thus, the functional significance of two forms of Rubisco activase is unknown. The primary structure of Rubisco activase is highly (>80%) conserved at the amino acid level with the exception of the C-terminal end of the longer form. There is at least one consensus nucleotide binding domain, the P-loop (Werneke et al. 1988b; Shen et al. 1991), which is involved in nucleotide-phosphate binding (Salvucci et al. 1993). Other regions involved in ATP binding and catalysis have been identified (Salvucci 1993, Salvucci and Klein 1994, Salvucci et al. 1994).

Occurrence Rubisco activase protein has been detected immunologically in all higher plant species that have been examined (Salvucci et al. 1987b), in two unicellular green algal species (Roesler and Ogren 1990; McKay et al. 1991) and, less-definitively, in cyanobacteria (Friedberg et al. 1993). In Chlamydomonas reinhardtii and Coleochaete scutata, Rubisco activase is localized in the pyrenoid along with most, if not all, of the Rubisco (McKay et al. 1991). In an interesting study of C3 and C4 Atriplex species, Hudson et al. (1992) showed that the spatial distribution of mRNA transcripts for Rubisco activase parallels the distribution of transcripts for the Rubisco small subunit. This result indicates that Rubisco activase like Rubisco is restricted to bundle sheath chloroplasts in C4 species. The restricted distribution of Rubisco activase to cell types or subcellular structures that contain Rubisco is consistent with the role of a specific modulator of Rubisco. The occurrence of Rubisco activase in Ca plants and unicellular green algae is quite revealing since in both groups the CO2 concentration at the site of Rubisco is elevated. The presence of Rubisco activase in plants that concentrate intercellular CO2 suggests that a high CO2 concentration at the active site of Rubisco is insufficient to promote carbamylation

without Rubisco activase. Apparently, the tight binding of RuBP to decarbamylated Rubisco presents an impediment to carbamylation even in species that possess mechanisms for concentrating intercellular CO2. A requirement for Rubisco activase in CO2-enriched environments is consistent with in vitro studies which show that RuBP inhibits Rubisco carbamylation even when the CO2 concentration is 20 times ambient (Portis et al. 1986).

The mechanism of Rubisco activase

The first measurements of Rubisco activase made use of chloroplast lysates to confirm the existence of an activase for Rubisco. In this simple system, the activity of endogenous Rubisco (i.e., the activation state) increased when lysates were illuminated in the presence of RuBP and an electron acceptor (Salvucci et al. 1985; Portis et al. 1986). Other experiments showed that a stromal protein missing from the rca mutant was the causative agent (Salvucci et al. 1985). Eventually, the requirement for thylakoids, light and electron acceptor was traced to a requirement for ATP and these components were replaced by ATP plus an ATP regenerating system (Streusand and Portis 1987). Further refinements to the system, including the use of purified recombinant Rubisco activase (Werneke et al. 1988a; Shen et al. 1991; Salvucci and Klein 1994), have made it possible to examine Rubisco activase in a system composed entirely of purified components.

Carbamylation in the presence of RuBP; the basis for assay of Rubisco activase As discussed above, tight-binding of RuBP to decarbamylated sites inhibits Rubisco activity by preventing carbamylation and promoting decarbamylation. When Rubisco activase and ATP are present, Rubisco activity is no longer inhibited by RuBP (Fig. 2). Werneke et al. (1988a) showed that there is an increase in carbamylation of Rubisco concomitant with the increase in Rubisco activity. Thus, Rubisco activase overcomes inhibition of enzyme activity by facilitating carbamylation of Rubisco in the presence of RuBP. Similarly, Rubisco activase also relieves inhibition caused by the tight binding of sugar-phosphates to earbamylated Rubisco. Evidence from Wang and Portis (1992) suggests that the major role of Rubisco activase is in pro-

moting dissociation "of RuBP and other tight-binding

sugar-phosphates from Rubisco, thus freeing the active site for activation by C02 and/or catalysis. Facilitated dissociation of RuBP from Rubisco appears to have the side-benefit of lowering the Kaet(CO2). At air-levels of CO2 and in the absence of RuBP, only about 20% of the active-sites are carbamylated (Portis et al. 1986). However, in the presence of Rubisco activase and RuBP the Kact(CO2)for carbamylation is reduced four-fold to about 4 pM (Portis et al. 1986). The lower CO2 requirement for activation supports a level of carbamylation at air-levels of CO2 equivalent to about 70-90% of the Rubisco sites. This level of carbamylation is sufficiently high to account for the observed rates of CO2 fixation at saturating irradiance. The ability to promote carbamylation of Rubisco in the presence of RuBP (i.e., to activate Rubisco) can be easily measured. In the two-stage assay, decarbamylated Rubisco previously complexed with RuBP is incubated with Rubisco activase in a buffered assay containing CO2, Mg 2+, RuBP, ATP and an ATP regenerating system (Fig. 3). The increase in carbamylation that occurs during incubation with Rubisco activase is measured indirectly in the second stage of the assay by determining Rubisco activity in aliquots of the mixture. Without Rubisco activase or ATP, the high level of RuBP present in the first stage of the assay prevents spontaneous carbamylation of Rubisco and the corresponding increase in enzyme activity (Fig. 3B). However, in the presence of Rubisco activase, Rubisco activity increases to a steady state level as carbamylation proceeds in the presence of RuBP.

mation of a pH gradient (Campbell and Ogren 1990, 1992, 1995). The ATPase activity of Rubisco activase appears to be essential for Rubisco activation. Non-hydrolyzable analogs of ATP and nucleotide triphosphates that do not bind or are not hydrolyzed by Rubisco activase do not support activation of Rubisco (Robinson and Portis 1989; Salvucci et al. 1993, 1994). ADP inhibits ATP hydrolysis and Rubisco activation to a similar extent and inactivation of Rubisco activase by high temperature (Robinson and Portis 1989), chaotropic agents or prolonged exposure to UV irradiation (Salvucci, unpublished) causes concomitant reductions in ATPase and Rubisco activation activities. Similarly, targetted inactivation of ATPase activity by chemical modification, photoaffinity labeling and site-directed mutagenesis also inactivates Rubisco activation (Shen et al. 1991; Salvucci 1993; Salvucci et al. 1993, 1994; Salvucci and Klein 1994) Although Rubisco activation requires hydrolysis of ATP by Rubisco activase, there does not appear to be a tight coupling between the two activities. For example, the rate of ATP hydrolysis by Rubisco activase is not affected by the presence of Rubisco and/or RuBP (Robinson and Portis 1989). Also, a sitedirected mutant in which the P-loop residue Glnl09 was changed to a Glu, had a higher specific activity for activation compared to the wild type, but a lower specific ATPase activity (Shen and Ogren 1992). The lack of coupling between ATP hydrolysis and Rubisco activation is an aspect of the Rubisco activase mechanism that has yet to be resolved.

ATP hydrolysis is essential for activation

Interaction of Rubisco activase with Rubisco

The ATP that is required for Rubisco activation is hydrolyzed by Rubisco activase, producing ADP and Pi (Robinson et al. 1988; Robinson and Portis 1989). ADP inhibits ATP hydrolysis and has a corresponding effect on Rubisco activation (Robinson and Portis 1989). In this way, activation of Rubisco by Rubisco activase is dependent on the ATP/ADP ratio (Fig. 2). This dependence partially explains the close relationship between Rubisco activation and the energization status of the chloroplast (Salvucci et al. 1987a; Robinson and Portis 1988), and thus accounts for the responsiveness of Rubisco activation to environmental factors. In addition, there is also evidence that Rubisco activase activity is promoted by light via a mechanism that is linked to electron flow through P S I and the for-

Rubisco activase 'activates' Rubisco by 1) facilitating carbamylation of Rubisco in the presence of RuBP and 2) relieving inhibition by tight binding inhibitors. Both of these activities reflect a general ability of Rubisco activase to promote dissociation of sugar-phosphates that bind to Rubisco. In each case, the substrate for Rubisco activase is a specific form of Rubisco, i.e., Rubisco that contains sugar-phosphate bound at the active site. Rubisco activase converts this form of Rubisco to a form that binds sugar-phosphates less tightly. The available evidence suggests that conversion of Rubisco from the tight to the loose binding form requires the binding and hydrolysis of ATP by Rubisco activase. Rubisco activase does not appear to covalently modify Rubisco or to metabolize sugar-phosphates

6

Rublsco activase assay

varlouo llm

I

-------

~___.,~_~

%0

D~'c-p~

A

~ fig

1 ~m~Trm~m~o¢

°' o

Time, (8)

B

Fig. 3. Assayfor Rubiscoactivaseactivity.A. Componentsand procedurefor the two stageassay. In the first stage, decarbamylatedRubisco complexed with RuBP is incubated with activase and the componentsshown in a buffered reaction. At various times, aliquots of the first stage mixture are removed for determination of Rubisco activity in a 30 s reaction. B. Time course of Rubisco activity after incubation of decarbamylatedRubiscocomplexedwith RuBP in the presence (complete)and absence(minus ATP and/oractivase)of Rubiscoaefivaseand ATE (Portis 1990). Instead, the interaction between Rubisco and Rubisco activase appears to involve a physical association between the proteins and a resultant conformational change in Rubisco. Indirect evidence for a physical association is the observation that Rubisco activase exhibits species selectivity. For example, Rubisco activase from Solanaceae species like tobacco, tomato and petunia are poor activators of Rubisco from non-Solanaceae species like spinach, Arabidopsis, pea and barley (Wang et al. 1992). Likewise, Rubisco activase from spinach promotes activation of Rubisco from non-Solanaceae species and even the green alga Chlamydomonas, but is a very poor activator of Solanaceae Rubisco. Interestingly, tobacco Rubisco activase inhibits activation of spinach Rubisco by spinach Rubisco activase and vice versa (Salvucci 1992). Also, an inactive mutant Rubisco activase, produced by directed mutagenesis of Lys-247, inhibits wild type Rubisco activase from activating Rubisco (Salvucci and Klein 1994). These observations suggest that subunit interactions between Rubisco and Rubisco activase are very specific and that proper interactions and stoichiometries are necessary for optimal activity. Chimeric proteins composed of regions of Rubisco activase from incompatible species (Esau et al. 1992) and a truncation mutant missing 50 amino acids from

the N-terminus (van de Loo and Salvucci 1995) have been used to determine the domains responsible for Rubisco activation and ATPase activity. The results show that regions at both the N- and C-terminus of the protein are involved in Rubisco activation. In particular, the N-terminus of Rubisco activase appears to be involved in Rubisco activation and is not required for ATP hydrolysis (van de Loo and Salvucci, unpublished).

Model for the mechanism of Rubisco activase A possible mechanism for Rubisco activase involving a physical association between Rubisco activase and Rubisco is presented in Fig. 4. The model shows a multimeric form of Rubisco activase containing bound ATP interacting with decarbamylated Rubisco containing RuBP at the active site. It is important to note that the Rubisco substrate can also be carbamylated, in which case the active site will be occupied by a tight-binding inhibitor. In either case, ATP hydrolysis by Rubisco activase causes global conformational changes that affect the active site of Rubisco, converting or isomerizing (Portis 1990) Rubisco into a form that binds sugar-phosphates less tightly. In the new conformation, tight-binding sugar-phosphates dissociate more readily from Rubisco, freeing the active site

Ru~:~lvlme ATP h ~ r o ~ .

Bct/vw~

I

Interaction I

conformMional changes

I

dluoolatlon e.~rbamyttnon RuBP

..% inactive Rubisco (decarbarayl~t)

activated Rubisco (carbam~

Model for the mechanism of Rubisco activase. In the model, a multimeric form of Rubisco activase (shaded figures) containing bound ATP interacts with decarbamylated Rubisco containing RuBP tightly bound at the active site. ATP hydrolysis causes conformational changes that alter the conformation around the Rubisco active site, thus lessening the binding for RuBE RuBP dissociates from the active site and the site is now free for carbamylation lay CO2. The model as shown depicts only the functional unit of Rubisco (i.e., a dimer of large subunits), but interaction between Rubisco activase and Rubisco may also involve the Rubisco small subunits. Fig. 4.

for spontaneous carbamylation by CO2 and/or catalysis upon binding RuBP (Fig. 4). To explain the lower Kaet(CO2) in the presence of Rubisco activase, the model shows that the conformation produced by the action of Rubisco activase has a higher affinity for carbamylation than the native enzyme. Andrews et al. (1995) have also proposed a model for the mechanism of Rubisco activase. Although their model was conceived independently of ours, it contains many of the same features and discusses in greater detail the possible structural changes of Rubisco that cause the tight- and loose-binding conformations. One small but important difference between the two models concerns the role of ATP hydrolysis. Andrews et al. (1995) propose that ATP hydrolysis occurs when Rubisco activase is not bound to Rubisco and that its function is to return Rubisco activase to a conformation that can interact with Rubisco. In contrast, we believe that binding of ATP promotes the interaction between Rubisco activase and Rubisco and that productive changes in Rubisco conformation occur when ATP is hydrolyzed by Rubisco activase complexed with Rubisco. Clearly, additional studies are needed to determine the precise role of ATP hydrolysis in the Rubisco activase mechanism. Thus far, attempts to isolate a stable binary complex of Rubisco activase and Rubisco have been unsuccessful. The only evidence for complex formation is between denatured Rubisco and native Rubisco activase (Jimenez et al. 1995). Nevertheless, the species specificity of Rubisco activase described above sug-

gests that the interaction between Rubisco activase and Rubisco involves a physical association. Specific interactions among Rubisco activase subunits also occur and these appear to be necessary for optimal activity. Studies with solvent excluding reagents like polyethylene glycol showed that Rubisco activase is a highly self-associating protein that exhibits enhanced enzymatic activity in the associated state (Salvucci 1992). The enhanced activity is characterized by an increased Vmax for ATP hydrolysis and Rubisco activation and an improved affinity for ATP and Rubisco. The tendency of Rubisco activase to aggregate also occurs at high concentrations of Rubisco activase protein (Salvucci 1992; Wang et al. 1993) or in the presence of Mg-ATP or Mg-ATPTS (Wang et al. 1993). Wang et al. (1993) have suggested that aggregated Rubisco activase is the functional form required for Rubisco activation. Self-association of Rubisco activase has made it difficult to define the molecular mass of the holoenzyme or the precise stoichiometry for the Rubisco activase-Rubisco interaction. In addition, it is not known if the active sites on each subunit function independently. Inhibition of Rubisco activation by the addition of mutant Rubisco activase to wild type enzyme suggests that the subunits of Rubisco activase act cooperatively to activate Rubisco (Salvucci and Klein 1994). In this way, Rubisco activase may be similar to the GroEL chaperonin which is highly cooperative at the level of the heptameric rings (Hayer-Hartl et al. 1995 and references therein). In fact, the recently proposed model for chaperonin-mediated protein fold-

ing, in which the two GroEL torroids are allostericaily connected in an ATP or ADP state, corresponding to low and high-affinity states for polypeptide binding (Hayer-Hartl et al. 1995), could also provide the basis for a mechanism for subunit interactions of Rubisco activase. Based on the concentration of Rubisco activase and Rubisco protein in leaves (Mate etal. 1993; Klein and Salvucci 1995), we calculate that there is approximately one Rubisco activase monomer per 8 to 24 Rubisco active sites in vivo. However, conditions in the chloroplast would promote self-association of Rubisco activase into higher ordered oligomers. As a tetramer, one Rubisco activase would be available to service 32 to 96 Rubisco active sites in normal leaves and as many as 1600 Rubisco active sites (i.e., 200 holoenzymes) when Rubisco activase levels are reduced by antisense constructs (Mate et al. 1993; Andrews et al. 1995).

Outstanding problems for future research Interaction with Rubisco In the broad sense of the definition, Rubisco activase appears to be a type of molecular chaperone, 'a protein that binds to and stabilizes an otherwise unstable conformer of another protein - and by controlled binding and release of the substrate protein, facilitates its correct fate in vivo: be it folding, oligomeric assembly, transport to a particular subcellular compartment, or controlled switching between active/inactive conformations' (Hendrick and Hartl 1993). The available evidence suggests that the main action of Rubisco activase is in 'switching' Rubisco to a conformation that binds sugar-bisphosphates less tightly. Like the GroE chaperonins (Hendrick and Hartl 1993), Rubisco activase hydrolyzes ATP during its interaction with Rubisco. However, unlike the GroE chaperonins, Rubisco activase interacts with the native (i.e., folded and assembled) form of Rubisco. This interaction between Rubisco and Rubisco activase almost certainly involves multiple turnovers in order to continually free Rubisco of 'fall-over' compounds and to counteract the tendency of Rubisco to decarbamylate and rebind RuBP. Probably the most important outstanding question concerning the mechanism of Rubisco activase is the nature of its interaction with Rubisco. At present, we assume that ATP hydrolysis causes conformational changes in Rubisco activase that affect the active site of Rubisco. However, no binary complex has been

isolated between Rubisco activase and native Rubisco and the domains involved in protein-protein interaction have not been identified. Similarly, there is little information about the changes in Rubisco conformation that alter the binding affinity for sugar-phosphates. Thus, fundamental details concerning the interaction of Rubisco activase and Rubisco remain to be elucidated.

Role of ATP A second important question concerning the mechanism of Rubisco activase is the role of ATP hydrolysis. Presumably, energy from ATP hydrolysis is used to induce conformational changes that affect the binding affinity of Rubisco for sugar-phosphates. Comparison of the rates of ATP hydrolysis and Rubisco activation show that about 10 moles of ATP are hydrolyzed for every mole of Rubisco active sites carbamylated. However, Rubisco activase catalyzes ATP hydrolysis in the absence of Rubisco and the rate of hydrolysis is not coupled to Rubisco activation (Robinson and Portis 1989; Salvucci, unpublished). This lack of coupling indicates that the relationship between ATP hydrolysis and conformational changes is rather subtle. In the GroEL/ES system, the ATPase activity of GroEL is also uncoupled unless GroES is present to couple it to the release of protein. Perhaps an as yet unidentified factor is required to couple Rubisco activation to ATP hydrolysis to avoid unregulated ATPase activity in vivo.

Regulation of Rubisco activase It is still not clear how Rubisco activase is regulated. The ATP/ADP is certainly an important determinant of Rubisco activase activity both in vitro and in isolated chloroplasts (Robinson and Portis 1989). However, in leaves the ATP/ADP ratio changes very little in response to irradiance. Since metabolite measurements determine total levels, it is possible that the changes in the pool size of free nucleotide are masked by compensatory changes in the pool of bound nucleotide. Also, the relative affinities for ADP and ATP are dependent on H + and Mg 2+ (Wang and Portis 1991), the concentrations of which change in the chloroplast in the light and dark (Heber et al. 1982). Alternatively, stimulation of Rubisco activase by electron transport through PS I (Campbell and Ogren 1990, 1992) or possible light activation of Rubisco activase (Lan et al. 1992) may provide mechanisms for regulating Rubisco activase

activity. Identification o f the components involved in the association o f Rubisco activase with the thylakoid membrane is a major area for future research.

The evolution and distribution of Rubisco activase Rubisco from photosynthetic bacteria has a lower specificity for CO2 and binds RuBP much less tightly than higher plant or green algal Rubiscos (Jordan and Ogren 1981; Jordan and Chollet 1983). These observations suggest that improvements in substrate specificity necessitated tighter binding o f RuBP to Rubisco. The tighter binding o f RuBP causes the complications that require Rubisco activase; i.e., inhibition o f carbamylation by RuBP and production o f 'fall-over' compounds. Rubisco activase apparently evolved as a mechanism to overcome the deleterious effects o f fight binding of sugar-phosphates. Evidence has recently been presented for the presence o f a Rubisco activase-like gene downstream from the Rubisco small subunit gene in the heterocystic cyanobacteriaAnabaena (Li et al. 1993). Like the rca gene in higher plants (Zielinski et al. 1989; MartinoCatt and Ort 1992; Pilgrim and McClung 1993; Klein and Salvucci 1995), the gene in cyanobacteria is controlled by light at the level of transcription (Li and Tabita 1994). The occurrence o f Rubisco activase in cyanobacteria is somewhat surprising considering that Rubisco in these organisms exists in an elevated CO2 environment and does not bind sugar-phosphates as tightly as the higher plant enzyme (Read and Tabita 1992 and references therein). Thus far, it is not known if the Rubisco activase-like gene in cyanobacteria encodes for a functional Rubisco activase protein since the deduced amino acid sequence is considerably different than higher plant and green algal Rubisco activase (Li et al. 1993). Further study o f the distribution of Rubisco activase in lower organisms may help to clarify the role o f Rubisco activase and its evolutionary origin.

Acknowledgements We would like to thank Dr T. John Andrews (Auslralian National University, Canberra) for kindly providing information in advance of publication.

No~s 1. Activationis defined in general terms as any increase in apparent specific activity. 2. The term 'fall-over' refers to the progressive inhibition or 'fallover' ofaclivity that accompaniesthe gradualformationof inhibitory compounds at the active site of Rubisco.

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