Liquid-Liquid Phase Transition Drives Intra-chloroplast Cargo Sorting.

Area of Science:

• Plant Biology
• Molecular Cell Biology
• Biochemistry

Background:

• Organelle biogenesis is essential for eukaryotic cell function.
• Chloroplasts possess complex internal membranes, and protein transport within them is not fully understood.
• Mechanisms for moving nuclear-encoded proteins to thylakoid membranes in chloroplasts require elucidation.

Purpose of the Study:

• To identify proteins involved in the sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes.
• To elucidate the mechanism by which these proteins mediate cargo sorting and translocation.
• To investigate the role of liquid-liquid phase separation in intra-chloroplast protein transport.

Main Methods:

• Yeast two-hybrid screening and co-immunoprecipitation to identify protein interactions.
• Biochemical assays to study protein complex formation and liquid-liquid phase separation.
• In vivo and in vitro experiments to analyze protein targeting and translocation.

Main Results:

• Two ankyrin-repeat proteins, STT1 and STT2, were identified as mediators of cpTat pathway protein sorting.
• STT1 and STT2 form a hetero-dimer, and their N-terminal regions bind cpTat substrates, inducing liquid-liquid phase separation.
• The multivalent STT complex and its interaction with Hcf106 are critical for reversing phase separation and facilitating cargo translocation.
• Phase-separated droplets of STT proteins act as novel compartments for intra-chloroplast cargo sorting.

Conclusions:

• STT1 and STT2 mediate the sorting of chloroplast proteins to thylakoid membranes through a novel liquid-liquid phase separation mechanism.
• This process is crucial for efficient protein transport and organelle biogenesis in chloroplasts.
• Liquid-liquid phase separation represents a conserved mechanism regulating protein sorting in organelles.