Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

Joshi, Jidnyasa; Mueller‐Cajar, Oliver; Tsai, Yi-Chin Candace; Hartl, F. Ulrich; Hayer‐Hartl, Manajit

doi:10.1074/jbc.m114.613091

Cited by 41 publications

(53 citation statements)

References 46 publications

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“…Another sub- stitution, T418A (Chlamydomonas RbcL numbering), which replaces the indole side-chain of Trp-418 with the smaller methyl side-chain of alanine, could eliminate its van der Waals contact with small subunit Leu-6, although, in the Chlamydomonas structure, Ala-418 is in contact with small subunit Trp-4. However, large-small subunit interactions are quite robust and malleable, evidenced from the myriad of engineered hybrid enzymes with evolutionarily distant large-small subunits that have been expressed in higher plants, algae and bacteria (van der Vies et al, 1986;Kanevski et al, 1999;Wang et al, 2001;Sharwood et al, 2008;Genkov et al, 2010;Ishikawa et al, 2011;Joshi et al, 2015). Similarly, we discovered that Chlamydomonas RbcS can assemble with Synechococcus RbcL in E. coli to form hexadecameric holoenzyme (data not shown).…”

Section: Discussionmentioning

confidence: 90%

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

2016

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

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Section: Discussionmentioning

confidence: 90%

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

2016

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“…In a recent study, co‐expression of the large subunit and RAF1 from Arabidopsis in tobacco chloroplasts has shown that Rubisco from higher plants prefers native RAF1 for efficient assembly (Whitney et al., ). On the other hand, the red‐type Rubisco from Rhodobacter sphaeroides was able to assemble in E. coli in the absence of extra assembly factors, and evidence indicated that a C‐terminal extension in its small subunit plays a similar role as RBCX to achieve the efficient assembly of the L 8 S 8 complex (Joshi et al., ). As a similar C‐terminal extension is also present in the small subunit of Griffithsia monilis (), its Rubisco might also be able to assemble in a heterologous environment in a similar manner.…”

Section: Discussionmentioning

confidence: 99%

“…Those findings indicated that the folding and assembly requirements of cyanobacterial Rubisco are not as strict as the Rubisco from higher plants and can be readily met in tobacco. An extra C‐terminal extension on the small subunit of the red‐type Rubisco from Rhodobacter sphaeroides has recently been observed to mimic the role of assembly factors, RBCX and RAF1, which were absent in organisms with red‐type Rubisco, suggesting that red‐type Rubisco may not require specific assembly factors (Joshi, Mueller‐Cajar, Tsai, Hartl, & Hayer‐Hartl, ). Therefore, we decided to reassess the expression of red‐type Rubisco in higher plants.…”

Section: Introductionmentioning

confidence: 99%

Red algal Rubisco fails to accumulate in transplastomic tobacco expressing Griffithsia monilis RbcL and RbcS genes

Lin

Hanson

2018

Plant Direct

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In C 3 plants, the carbon fixation step catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) represents a major rate-limiting step due to the competing oxygenation reaction, which leads to the energy-intensive photorespiration and lowers the overall photosynthetic efficiency. Hence, there is great biotechnological interest in replacing the Rubisco in C 3 crops with a more efficient enzyme. The Rubisco enzymes from red algae are among the most attractive choices due to their remarkable preference for carboxylation over oxygenation reaction. However, the biogenesis of Rubisco is extremely complex. The Rubisco enzymes in plants, algae, and cyanobacteria are made up of eight large and eight small subunits. The folding of the large subunits and the assembly of the large subunits with the small subunits to form a functional holoenzyme require specific chaperonin complexes and assembly factors. As a result, previous success in expressing foreign Rubisco in plants has been limited to Rubisco large subunits from closely related plant species and simpler bacterial enzymes. In our previous work, we successfully replaced the Rubisco in tobacco with a cyanobacterial enzyme, which was able to support the phototrophic growth of the transgenic plants. In this work, we used the same approach to express the Rubisco subunits from the red alga Griffithsia monilis in tobacco chloroplasts in the absence of the tobacco Rubisco large subunit.Although the red algal Rubisco genes are being transcribed in tobacco chloroplasts, the transgenic plants lack functional Rubisco and can only grow in a medium containing sucrose. Our results suggest that co-expression of compatible chaperones will be necessary for successful assembly of red algal Rubisco in plants. K E Y W O R D Schloroplast transformation, Griffithsia monilis, photosynthesis, Rubisco

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“…This b-subunit is then on standby until the unfolded freshly synthesized nuclear-encoded a-subunits are translocated across five membranes [18] to bind to the bsubunit in the stroma, thereby triggering the folding of the asubunit and the subsequent formation of the quaternary structure.T his scenario bears some resemblance to the assembly of Ribulose-1,5-bisphosphate carboxylate/oxygenase (Rubisco), whereby chaperones fold and then keep the plastid-encoded Rubisco large subunits (RbcL) from aggregating until the Rubisco small subunit (RbcS) becomes available. [23][24][25] This study has potential to inspire improvements in biotechnology methods for making quaternary protein complexes in vitro,a nd additionally in understanding ultrafast light-harvesting and protein folding in the quantum and chemical biology fields,respectively.Based on the mechanism proposed here,itshould be possible to design and synthesize novel hybrid quaternary light-harvesting protein complexes through complementary (cooperative) interactions between otherwise unrelated subunits.T he lessons learned here could be extended to synthetic (macro)molecular systems guiding the construction of complex multimeric (quaternary) foldamers. [26] Overall, our results suggest that subunit self-chaperoning [10,11] is important for the folding of the cryptophyte lightharvesting complexes,and may be awidespread feature of the folding of heteromeric protein complexes.…”

Section: Angewandte Chemiementioning

confidence: 99%

Cooperative Subunit Refolding of a Light‐Harvesting Protein through a Self‐Chaperone Mechanism

et al. 2017

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The fold of ap rotein is encoded by its amino acid sequence,b ut how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear.H ere we show that two structurally different phycobiliproteins refold and reassemble in ac ooperative manner from their unfolded polypeptide subunits,w ithout biological chaperones.R efolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopyt op robe internal chromophores as am arker of quaternary structure.O ur results demonstrate ac ooperative,s elfchaperone refolding mechanism, whereby the b-subunits independently refold, therebyt emplating the folding of the asubunits,w hich then chaperone the assembly of the native complex, quantitatively returning all coherences.O ur results indicate that subunit self-chaperoning is ar obust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: http://dx.

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Role of Small Subunit in Mediating Assembly of Red-type Form I Rubisco

Cited by 41 publications

References 46 publications

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

Red algal Rubisco fails to accumulate in transplastomic tobacco expressing Griffithsia monilis RbcL and RbcS genes

Cooperative Subunit Refolding of a Light‐Harvesting Protein through a Self‐Chaperone Mechanism

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