The BSD2 ortholog in Chlamydomonas reinhardtii is a polysome‐associated chaperone that co‐migrates on sucrose gradients with the rbcL transcript encoding the Rubisco large subunit

Abstract

SUMMARYThe expression of the CO 2 -fixation enzyme ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which is affected by light, involves the cysteine-rich protein bundle-sheath defective-2 (BSD2) that was originally identified in maize bundle-sheath cells. We identified the BSD2 ortholog in Chlamydomonas reinhardtii as a small protein (17 kDa) localized to the chloroplast. The algal BSD2-ortholog contains four CXXCXGXG DnaJlike elements, but lacks the other conserved domains of DnaJ. BSD2 co-migrated with the rbcL transcript on heavy polysomes, and both BSD2 and rbcL mRNA shifted to the lighter fractions under oxidizing conditions that repress the translation of the Rubisco large subunit (RbcL). This profile of co-migration supports the possibility that BSD2 is required for the de novo synthesis of RbcL. Furthermore, BSD2 co-migrated with the rbcL transcript in a C. reinhardtii premature-termination mutant that encodes the first 60 amino acids of RbcL. In both strains, BSD2 shared its migration profile with the rbcL transcript but not with psbA mRNA. The chaperone activity of BSD2 was exemplified by its ability to prevent the aggregation of both citrate synthase (CS) and RbcL in vitro following their chemical denaturation. This activity did not depend on the presence of the thiol groups on BSD2. In contrast, the activity of BSD2 in preventing the precipitation of reduced b-chains in vitro in the insulin turbidity assay was thiol-dependent. We conclude that BSD2 combines a chaperone 'holdase' function with the ability to interact with free thiols, with both activities being required to protect newly synthesized RbcL chains.

The Rubisco Chaperone BSD2 May Regulate Chloroplast Coverage in Maize Bundle Sheath Cells

“…Subsequent work in maize showed that BSD2 likely interacts with nascent LS and/or newly imported small subunit as well as other assembly factors (RAF1 and RAF2) to assemble the Rubisco holoenzyme (Feiz et al, 2012(Feiz et al, , 2014. More limited data are consistent with analogous roles in Nicotiana benthamiana (Wostrikoff and Stern, 2007) and Chlamydomonas reinhardtii (Doron et al, 2014).…”

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

“…Escherichia coli DnaK/DnaJ or GroEL/GroES systems may additionally mimic specialized cyanobacterial (but not eukaryotic) Rubisco-interacting proteins at pre-assembly Rubisco biogenesis stages. The existence of such particular factors as interacting with RbcL nascent chain Bsd2 (Doron et al 2014) or specialized Cpn60 subunits required for rice RbcL folding (Kim et al 2013) has been confirmed for eukaryotic cells.…”

“…It is postulated that to achieve a proper tertiary structure RbcL requires the action of chaperonin (chloroplast Cpn60/Cpn20/Cpn10 or prokaryotic GroEL/GroES; Barraclough and Ellis 1980; Goloubinoff et al 1989). Moreover, before that stage, a specific DnaJ-like factor—Bsd2, found in eukaryotic chloroplasts seems to be crucial during the folding of the N-terminal domain of the large Rubisco subunit (Brutnell et al 1999; Doron et al 2014). To form the octameric core of Rubisco, properly folded RbcLs first need to be dimerized, and to achieve this goal the participation of the so-called assembly chaperones is required.…”

Engineering chloroplasts to improve Rubisco catalysis: prospects for translating improvements into food and fiber crops

“…The function of BSDII in plant chloroplasts is uncertain. BSDII may interact with newly synthesized L-peptides (Doron et al, 2014) or at the post-chaperonin stage for Rubisco subunit assembly (Feiz et al, 2014). See text for further details.…”