Cell-free reconstitution of peroxisomal matrix protein import using Xenopus egg extract

Abstract

Summary: Peroxisomes are vital metabolic organelles whose matrix enzymes are imported from the cytosol in a folded state by the soluble receptor PEX5. The import mechanism has been challenging to decipher because of the lack of suitable in vitro systems. Here, we present a protocol for reconstituting matrix protein import using Xenopus egg extract. We describe how extract is prepared, how to replace endogenous PEX5 with recombinant versions, and how to perform and interpret a peroxisomal import reaction using a fluorescent cargo.For complete details on the use and execution of this protocol, please refer to Skowyra and Rapoport (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cell separation/fractionation
• Metabolism
• Microscopy
• Model Organisms
• Molecular Biology