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Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplasts
R M Mould1, A Kapazoglou, J C Gray
1Department of Plant Sciences, University of Cambridge, UK. Ruth.Mould@plantsci.cam.ac.uk
Insights
Cytochrome f assembly into the cytochrome b6f complex does not require heme binding but depends on its C-terminal region. This C-terminal region is crucial for stabilizing the protein within the thylakoid membrane.
Area of Science:
- Plant molecular biology
- Photosynthesis research
- Protein assembly mechanisms
Background:
- The cytochrome b6f complex is essential for photosynthesis.
- Cytochrome f is a key component of this complex.
- Understanding cytochrome f's role in assembly is crucial for photosynthesis research.
Purpose of the Study:
- To investigate the structural requirements for cytochrome f assembly into the cytochrome b6f complex.
- To determine the necessity of heme binding for assembly.
- To identify the role of the C-terminal region in membrane integration and stability.
Main Methods:
- Import of radiolabeled chimeric precursor proteins into isolated pea chloroplasts.
- Analysis of thylakoid membrane protein complexes using nondenaturing gel electrophoresis.
- Site-directed mutagenesis to alter heme-binding sites and C-terminal residues.
Main Results:
- Cytochrome f mutants lacking heme-binding capacity were successfully assembled into the complex, indicating heme is not essential for assembly.
- A truncated cytochrome f lacking its C-terminal 33 amino acids was translocated but not assembled, highlighting the C-terminal region's importance.
- Mutant cytochrome f lacking both heme and the C-terminal region was unstable, suggesting proteolytic degradation in the absence of the membrane anchor.
Conclusions:
- Covalent heme binding is not required for the assembly of cytochrome f into the cytochrome b6f complex.
- The C-terminal region of cytochrome f, including its transmembrane anchor, is critical for proper assembly and stability within the thylakoid membrane.
- The absence of the C-terminal anchor exposes heme-less cytochrome f to proteolytic degradation.
Abstract:
Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of (35)S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system.