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STAR Protoc
2023 Mar 17;41:102111. doi: 10.1016/j.xpro.2023.102111.
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Cell-free reconstitution of peroxisomal matrix protein import using Xenopus egg extract.
Skowyra ML
,
Rapoport TA
.
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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.
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