16.16
Protein Transport to the Stroma
Chloroplasts are triple membrane structures with an outer membrane, an inner membrane, and a thylakoid membrane, each containing distinct metabolite t…
Most chloroplast proteins are synthesized in the cytosol and translocated as unfolded precursors.
These precursors contain a cleavable transit signal at the N-terminal and an internal import signal that targets the protein to different subcompartments.
Cytosolic chaperones interact with the transit signal and direct the precursor to the chloroplast outer membrane.
Multimeric complexes embedded in the chloroplast membranes called the translocase of the outer chloroplast membrane or TOC complex, and the translocase of the inner chloroplast membrane or TIC complex mediate protein transport to the internal subcompartments.
GTP-bound TOC receptors recognize and bind the transit signal. GTP hydrolysis allows the precursor to enter and pass through the TOC channel and move into the intermembrane space.
Next, the TIC complex allows the precursor to translocate into the stroma.
Stromal Hsp70 uses energy from ATP hydrolysis to pull the emerging peptide out of the TIC complex.
Once the complete peptide is released into the stroma, stromal processing peptidases cleave the transit signal releasing the active protein.
View the full transcript and gain access to JoVE Core videos
Q1: How do chloroplast proteins reach the stroma after synthesis?
Chloroplast proteins are synthesized in the cytosol as unfolded precursors containing a cleavable transit signal. Cytosolic chaperones direct these precursors to the chloroplast outer membrane, where the TOC complex recognizes the transit signal. The TIC complex then mediates translocation across the inner membrane into the stroma, where stromal Hsp70 pulls the emerging peptide through using ATP energy.
Q2: What role do transit peptides play in chloroplast protein targeting?
Transit peptides are cleavable N-terminal sequences of 13 to 146 amino acids that direct chloroplast precursor proteins to their correct destinations. These peptides are recognized by GTPases TOC159 and TOC34 at the chloroplast outer membrane. Once the protein enters the stroma, stromal processing peptidases cleave the transit signal, releasing the active protein.
Q3: How do the TOC and TIC complexes work together during protein import?
The TOC complex embedded in the outer chloroplast membrane recognizes and binds the transit signal through GTP-bound receptors. GTP hydrolysis allows the precursor to pass through the TOC channel into the intermembrane space. The TIC complex then facilitates translocation across the inner membrane, with TIC40 stimulating Hsp93 to use ATP energy and pull the precursor completely into the stroma.
Q4: What is the function of cytosolic chaperones in chloroplast protein transport?
Cytosolic chaperones Hsp90 and HOP bind newly synthesized chloroplast precursors and use ATP hydrolysis energy to maintain them in an unfolded state. These chaperones interact with the transit signal and direct the unfolded precursor to the chloroplast outer membrane, preparing it for recognition by TOC receptors and subsequent translocation.
Q5: How does stromal Hsp70 facilitate protein translocation into the stroma?
Stromal Hsp70 uses energy from ATP hydrolysis to pull the emerging peptide out of the TIC complex as it translocates across the inner chloroplast membrane. This active pulling mechanism helps move the complete precursor into the stroma. Once fully released, stromal processing peptidases cleave the transit signal, and Cpn60 chaperones fold the processed protein into its active conformation.
Q6: What happens to the transit peptide after the protein enters the stroma?
Once the complete precursor peptide is released into the stroma, stromal processing peptidases recognize and cleave the transit signal sequence from the N-terminus. This cleavage removes the targeting information and releases the mature, active protein. The processed protein is then folded by Cpn60 chaperones to achieve its functional three-dimensional structure.
Q7: Why is ATP hydrolysis essential for chloroplast protein import?
ATP hydrolysis provides energy at multiple steps of chloroplast protein import. Cytosolic chaperones use ATP to keep precursors unfolded, while stromal Hsp93 and Hsp70 use ATP energy to pull the emerging peptide through the TIC complex into the stroma. Sufficient ATP concentration coupled with proper temperature ensures efficient translocation across both the outer and inner chloroplast membranes.