Cardiolipin liposomes sequester a reactivatable partially folded rhodanese intermediate

Area of Science:

• Mitochondrial biochemistry
• Protein-lipid interactions
• Enzyme kinetics

Background:

• Mitochondrial protein import is crucial for cellular function.
• Cardiolipin (CL) is a key mitochondrial lipid involved in protein translocation.
• The enzyme thiosulfate sulfurtransferase plays a role in mitochondrial processes.

Purpose of the Study:

• To investigate the interaction between thiosulfate sulfurtransferase and different liposomes (CL, PtdSer, PtdCho).
• To determine the effect of liposomes on enzyme activity and folding state.
• To elucidate the mechanism of enzyme reactivation by detergents.

Main Methods:

• Preparation of large unilamellar vesicles (LUV) from various lipids.
• Binding assays to quantify enzyme-liposome interactions.
• Enzyme activity assays before and after incubation with liposomes.
• Protease digestion and fluorescence spectroscopy to analyze protein conformation.

Main Results:

• CL/LUV showed significantly higher binding of partially folded thiosulfate sulfurtransferase compared to PtdSer/LUV and PtdCho/LUV.
• Enzyme bound to CL/LUV was inactive but could be reactivated (~56%) with detergents and other agents.
• Protease digestion and fluorescence spectroscopy indicated enzyme was a folding intermediate when bound to CL/LUV and PtdSer/LUV.

Conclusions:

• Cardiolipin (CL) binds thiosulfate sulfurtransferase, sequestering it in a translocation-competent, partially folded state.
• Detergents likely facilitate enzyme reactivation by disrupting liposomes, allowing protein refolding.
• These findings support a role for CL in mitochondrial protein translocation and proper enzyme folding.