Structure and mechanism of the Rubisco-assembly chaperone Raf1

Abstract

Biogenesis of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits, requires assembly chaperones. Here we analyzed the role of Rubisco accumulation factor1 (Raf1), a dimer of ∼40-kDa subunits. We find that Raf1 from Synechococcus elongatus acts downstream of chaperonin-assisted RbcL folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by RbcS results in holoenzyme formation. Crystal structures show that Raf1 from Arabidopsis thaliana consists of a β-sheet dimerization domain and a flexibly linked α-helical domain. Chemical cross-linking and EM reconstruction indicate that the β-domains bind along the equator of each RbcL2 unit, and the α-helical domains embrace the top and bottom edges of RbcL2. Raf1 fulfills a role similar to that of the assembly chaperone RbcX, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.

Is RAF1 protein from Synechocystis sp. PCC 6803 really needed in the cyanobacterial Rubisco assembly process?

“…Cell extracts with confirmed soluble RbcL presence were additionally separated under non-denaturing conditions and probed with anti-RbcL or anti-RAF1 antibodies. Synechocystis RAF1 promoted folding of its own RbcL resulting in formation of a complex exhibiting a migration pattern similar to those reported for T. elongatus (Kolesinski et al 2014), or S. elongatus PCC 7942 (Hauser et al 2015) RbcL 2 -RAF1 2 (Fig. 6b).…”

“…In addition, it effectively showed the same function toward RbcLs from two other cyanobacteria ( T. elongatus and S. elongatus PCC 7942). However, in these cases only larger complexes resembling those identified previously as RbcL 8 –RAF 8 (Hauser et al 2015) were observed. Interestingly, spontaneous formation of a presumed octamer was observed for RbcL from T. elongatus alone.…”

“…The central cleft of RbcX recognizes a specific sequence in the C-terminal tail of one RbcL subunit, whereas its apical part provides an additional waypoint for appropriate positioning of the second RbcL subunit (Saschenbrecker et al 2007; Bracher et al 2011). RAF1, on the other hand, binds along the RbcL dimer interface, which suggests an alternate way of guiding the arrangement of bilateral large Rubisco subunits (Hauser et al 2015). Recently, another Rubisco assembly chaperone—RAF2, has been identified.…”

“…During biosynthesis of plant Rubisco, the assembly machinery instead depends on the RAF1 protein, which is confirmed by the lethality of RAF1-encoding gene disruption in maize (Feiz et al 2012) as well as the requirement for Arabidopsis thaliana RAF1 for proper biogenesis of Arabidopsis enzymes in tobacco cells (Whitney et al 2015). Although the ability of RAF1 to promote prokaryotic RbcL assembly in vitro as well as in Escherichia coli is indisputable (Hauser et al 2015; Kolesinski et al 2014), the physiological function of this factor in cyanobacterial cells remains unclear.…”

Surveying Rubisco diversity and temperature response to improve crop photosynthetic efficiency

“…However, advances in engineering precise changes in model systems continue to provide important developments that are increasing our understanding of Rubisco catalysis (Spreitzer et al, 2005;Whitney et al, 2011aWhitney et al, , 2011bMorita et al, 2014;Wilson et al, 2016), regulation (Andralojc et al, 2012;Carmo-Silva and Salvucci, 2013;Bracher et al, 2015), and biogenesis (Saschenbrecker et al, 2007;Whitney and Sharwood, 2008;Lin et al, 2014b;Hauser et al, 2015;Whitney et al, 2015).…”