FASE II: Planificación Específica y Ejecución

Assembly of the Rubisco enzyme has been studied in a variety o f different

experimental systems, including assembly in whole leaves (Roy et al., 1978; 1979), intact

isolated chloroplasts (Blair & Ellis, 1973; Barraclough & Ellis, 1980) and chloroplast extracts (Milos & Roy, 1984). Reconstitution of Rubisco assembly has been studied in

isolated yeast mitochondria (Hurt et al., 1986), in E.coli (reviewed in Section 1.4) and in

vitro (Voordouw et al., 1984). This section discusses a number of results obtained from these systems which have contributed to the current model of Rubisco asembly as presented in Figure 4.

The Rubisco small subunit is synthesized as a precursor by the cytosolic ribosomes in eukaryotic cells (see Section 1.2.3) and transported across the two envelope membranes. This transport occurs post-translationally since it has been shown that subunit precursors are

Figure 4. Synthesis an d assem bly of hexadecam eric Rubisco

Panel A shows a suggested pathway for the synthesis and assembly of Rubisco from plants. Rubisco large subunits are synthesized from chloropast genes (ctDNA) whereas Rubisco small subunits and the binding protein subunits are synthesized from nuclear genes (nDNA). The symbols P20 and P62 refer to the apparent molecular masses x 10*3 for the precursor polypeptides respectively. The wavy line indicates the role of light in the stimulation of the small subunit gene transcription. This diagram was taken from a paper by Ellis (1987). Panel B shows the proposed order of events in the formation of the hexedecameric Rubisco enzyme: 1. folding of the large subunit (L) monomer; 2. formation of the large subunit dimer (L2); 3. tertamerization of large subunit dimers to give the octameric (L8) core; 4. folding of the small subunit (S) monomer; 5. association of the small subunits with the L8 core to give

the hexedecameric holoenzyme. This diagram was adapted from a paper by Goloubinoff et

al. (1989)

3 9 / 1

A

Schmidt, 1978) and from Piswn sativum (Highfield & Ellis, 1978). These observations are in agreement with the failure to find cytosolic ribosomes bound to the chloroplast envelope during chloroplast development (Ellis, 1981). This type of protein transport thus differs from the observed co-translational translocation of secretory proteins across the membrane of the endoplasmic reticlulum, which is dependent on concomitant protein synthesis

(reviewed by Walter et al., 1984)

The small subunit precursors bind to the outer surface of isolated intact chloroplasts. When the chloroplasts are pretreated with proteases the subsequent uptake of small subunit

precursors is inhibited (Chua & Schmidt, 1978; Cline et al., 1985), suggesting that

membrane receptor proteins probably interact with the precursor polypeptides. Precursors which are bound to isolated envelope membranes were shown to be sensitive to low

concentrations of trypsin (Pfisterer et al., 1982). The sensitivity of proteins to proteases is

often used as a measure for the folded state of the protein and the protease-sensitivity of the bound precursor molecules thus suggesting a partially folded conformation.

Grossman et al. (1980) showed that energy in the form of ATP is required to move

the precursors across the chloroplast membranes. Either during or after transport the small subunit precursor is processed to its mature size by a highly specific protease located in the

stromal fraction (Dobberstein et al., 1977; Smith & Ellis, 1979). The processing of the 20

kDa small subunit precursor from Pisum sativum to the 14 kDa mature polypeptide is

independent of ATP and proceeds in two steps. Robinson & Ellis (1984) observed a 18 kDa intermediate form when small subunit precursors were treated with the partially purified stromal protease. The significance of this two-step processing is unknown. It has recently been shown that import o f the small subunit precursor into isolated intact chloroplast requires a chloroplast ATPase (Pain & Blobel, 1987). The function of this ATPase is unknown at present since it does not generate a membrane potential which could be utilized to drive import. The small subunit presequence which is thought to contain the information required for transport into chloroplast, has successfully been used to target foreign bacterial

proteins into chloroplasts both in vivo and in vitro (recently reviewed by Keegstra, 1989).

Although the synthesis of the Rubisco subunits and the transport of the small subunit precursor into chloroplasts are well-studied processes (Fig. 4A), the mechanism by which the two types of subunits assemble into a hexadecameric protein complex which is biologically functional is poorly understood. The available evidence suggests a complex pathway for Rubisco asssembly. The observations that newly-synthesized Rubisco large subunits assemble into holoenzyme molecules in isolated intact chloroplasts (Barraclough & Ellis, 1980) suggests the existence of a pool of assembly-competent free small subunits. The possibility that the newly-synthesized large subunits either assemble with small subunits that have been released from pre-existing Rubisco enzyme complexes or exchange with large subunits within these complexes is unlikely but cannot be ruled out. Pools of free cytosolically synthesized small subunits have also been identified in extracts of leaves of

Pisum sativum (Roy et al., 1978; 1979). Unassembled small subunits are rapidly degraded in Chlamydomonas reinhardii (Schmidt & Mishkind, 1983), and have a half-life of less than 7.5 min, which suggests that the pool of unassembled subunits is probably small, but

sufficient to permit Rubisco assembly for a short time in vitro.

Barraclough & Ellis (1980) were the first to show that in intact chloroplasts isolated

from Pisum sativum, the newly-synthesized large subunits bind to another abundant stromal

protein which they termed the Rubisco large subunit binding protein. Analysis of the newly-

synthesized large subunits from either P.sativum seedlings or isolated intact chloroplasts on

sucrose density gradients showed that they sediment as tw o distinct forms with

sedimentation values quoted as 7S and 29S (Roy et al., 1982). The 7S complex has been

suggested to represent large subunit dimers, while the 29S complex is identical to the Rubisco large subunit binding protein complex identified by Barraclough and Ellis (1980).

The binding of the large subunits to this binding protein also occurs in vivo (Ellis, 1981),

and time course experiments with isolated intact chloroplasts indicated that the large subunits bind to this protein prior to assembly into the holoenzyme. Barraclough & Ellis (1980) proposed that this association may be an intermediate step in the pathway of Rubisco assembly. The properties of the Rubisco large subunit binding protein and its possible role in assembly of the Rubisco holoenzyme are reviewed in Section 1.3.

The demonstration that bacterial proteins can be imported into isolated intact chloroplasts providing that they contain the Rubisco small subunit presequence at the

aminoterminus (reviewed by Cashmore et al., 1985), caused Hurt et al. (1986) to use a

similar approach to investigate import of chloroplast protein into intact mitochondria isolated

from Saccharomyces cerevisiae. When the presequence of cytochrome c oxidase subunit IV

(a protein normally transported into mitochondria) was fused to the aminoterminus of the

Rubisco large and mature small subunit from Chlamydomonas reinhardii, these fusion

proteins were efficiently imported into isolated intact yeast mitochondria. In mitochondria containing both processed Rubisco large and small subunits, no formation of oligomeric protein complexes was observed as judged by migration on sucrose density gradients (Hurt

etal. ,1986). Since mitochondria normally do not contain Rubisco the mitochondrial matrix may not be a suitable environment for assembly, and the failure to assemble the Rubisco subunits may indicate the requirement for the chloroplast Rubisco large subunit binding protein. This idea is supported by the recent observation that cyanobacterial Rubisco large subunits assemble into holoenzme oligomers after being imported as fusion proteins containing the soybean Rubisco small subunit presequence into intact chloroplasts isolated

from P. sativum (Gatenby etal., 1988).

A traditional means to investigate the folding and assembly of proteins is to study the

dissociation and reassociation of purified protein in vitro. Treatment of the hexadecameric

Rubisco purified from Spinacia oleracea with a low concentration of urea (3-4M) causes the

small subunits to unfold and dissociate from the oligomeric protein complex with

proportional loss of enzyme activity (Voordouw et al., 1984). The sedimentation coefficient

of the remaining protein structure Cs20,w=15.0 S) indicates a conformation close to an octamer, possibly with reduced stability. The reconstitution of an enzymically active enzyme by removing the urea either in the presence or the absence o f the small subunits has been unsuccessful because the large subunits aggregate and form insoluble complexes (Voordouw

et al-, 1984). The Rubisco purified from the cyanobacteria Synechococcus and Prochloron is a hexadecameric enzyme from which small subunits can be isolated in monomeric form after

large subunits are stable octamers which retained the ability to assemble with either

homologous or heterologous small subunits into functional hexadecamers (Andrews et al.,

1984; Andrews & Lorimer, 1985; Incharoensakdi et al., 1985). Assembly of a hybrid

Rubisco from its subunits synthesized in E.coli has also been reported (see Section 1.4.3.;

van der Vies et al., 1986).

Since the dissociation of the small subunits from the S.oleracea holoenzyme leaves

the large subunits in a loose and flexible octameric form (Voordouw et al., 1984), this may

allow inter and/or intra specific interactions to occur more frequently than in the assembled holoenzyme. Some of these interactions may lead to new conformations of the large subunits that are no longer capable of binding small subunits and are no longer soluble in aqueous

solution. Thus it seems likely that reduction of the flexibility of the S.oleracea large subunit

octamer will decrease the frequency of inter and/or intra specific interactions and hence may pevent aggregates to be formed. This idea is supported by the recent observation that when

Rubisco purified from N.tabacum is immobilized on Sepharose 4B beads the loss of

enzymic activity caused by the urea-dependent dissociation of the small subunits is fully restored when the concentration of urea is reduced to 0.5M in the presence of isolated small

subunits (Liren et al., 1988). These observations indicate the requirement for a factor which

stabilizes the large subunit octamers. The stabilization of the large subunits and/or assistance

in the assembly process in vivo, is a function which has been proposed for the Rubisco large

subunit binding protein (Barraclough & Ellis, 1980).