16.17
Protein Transport to the Outer Chloroplast Membrane
Chloroplast outer membrane proteins encoded by the nucleus are synthesized in the cytosol. Soon after synthesis, they bind cytosolic factors such as 1…
Chloroplast protein precursors targeted to the outer membrane carry non-cleavable transit signals at their N-terminal end. Precursor proteins dock onto the TOC complex and translocate across the outer membrane.
The TOC complex interacts with the TIC complex of the inner membrane and the TIC stromal components, forming a TOC-TIC super complex that helps the proteins move inwards.
As the precursor enters the TIC complex, a polyglycine stretch close to the transit signal stalls further translocation, arresting the precursor at the TOC-TIC super complex.
Plastidic type 1 signal peptidases cleave the polyglycine stretch while an N-terminal domain of the TOC complex, called the polypeptide transport-associated domain or POTRA domain, functions as a chaperone preventing precursor aggregation.
The processed precursor is then transferred to an insertion pore complex called the outer membrane protein for integration into the chloroplast outer membrane.
View the full transcript and gain access to JoVE Core videos
Q1: What are transit signals and how do they direct chloroplast proteins to the outer membrane?
Transit signals are non-cleavable N-terminal sequences on chloroplast protein precursors that direct them to the outer membrane. These signals are recognized by TOC receptors in the cytosol and guide precursor docking onto the TOC complex. The transit signal remains attached throughout translocation across the outer membrane, enabling proper protein targeting and insertion.
Q2: How do the TOC and TIC complexes work together during chloroplast protein import?
The TOC complex on the outer membrane and TIC complex on the inner membrane interact to form a TOC-TIC super complex that facilitates protein translocation. This super complex creates a continuous channel allowing precursors to move from the cytosol through both membranes. The coordinated action of these complexes ensures efficient and directional protein transport into the chloroplast.
Q3: What role does the polyglycine stretch play in regulating protein translocation?
A polyglycine stretch located near the transit signal acts as a translocation brake, stalling the precursor at the TOC-TIC super complex. This arrest prevents the protein from entering the stroma prematurely. Plastidic type 1 signal peptidases then cleave this polyglycine stretch, allowing the processed precursor to proceed to the outer membrane insertion machinery.
Q4: What is the POTRA domain and how does it function during protein translocation?
The POTRA domain is an N-terminal region of the TOC complex that functions as a molecular chaperone. It prevents precursor aggregation and maintains proteins in an unfolded state during translocation across the outer membrane. This chaperone activity is essential for successful protein import and proper targeting to the chloroplast.
Q5: How do cytosolic chaperones prepare chloroplast precursor proteins for translocation?
Cytosolic factors including 14-3-3 protein and Hsp70 chaperones bind to newly synthesized chloroplast precursors immediately after synthesis. These chaperones maintain precursors in an unfolded state, preventing premature aggregation and ensuring they remain competent for translocation. This preparation is critical before precursors encounter the TOC complex at the chloroplast membrane.
Q6: What are the two proposed models for how TOC receptors recognize and bind precursor proteins?
Model 1 proposes TOC159 binds the precursor first, then docks via TOC34, triggering GTP hydrolysis and translocation. Model 2 suggests TOC34 is the initial receptor, binding a phosphorylated transit signal, dephosphorylating it, and transferring the precursor to TOC159. Both models involve GTP hydrolysis to drive precursor movement through the TOC complex into the intermembrane space.
Q7: How are processed chloroplast outer membrane proteins inserted after translocation?
After plastidic type 1 peptidases cleave the polyglycine signal in the intermembrane space, the processed precursor is transferred to an insertion protein called outer membrane protein (OMP). The OMP complex folds and integrates the processed protein into the chloroplast outer membrane, completing the targeting and insertion pathway.