Characterization of Pex3 and Pex16 Trafficking During De Novo Peroxisome Biogenesis

Abstract

Peroxisomes can be formed de novo or through growth and division. The de novo formation pathway represents a novel form of protein trafficking as it involves the fusion of vesicles originated from two distinct compartments: the endoplasmic reticulum (ER) and the mitochondria. Delineating the molecular mechanisms behind the trafficking events is of great interest to the field.

In this study, we employed the Retention Using Selective Hooks (RUSH) to study the trafficking of these two vesicles. For the mitochondria part, we designed a mitochondrial hook with a mitochondria-localizing monoamine oxidase (MAO) to control the release of the peroxin, Pex3, from the mitochondrial outer membrane (MOM). Testing of the construct with non-mitochondrial resident viral glycoprotein VSVG locked the cargo as vesicle tethers on the MOM and released the cargo to its conventional trafficking route upon biotin addition. Implementing the mitochondrial RUSH (M-RUSH) system on Pex3 revealed a dual localization of the cargo of mitochondria and peroxisomes in wild-type HeLa cells, with both entities being released 24 hours after biotin release. Rescue using the M-RUSH in peroxisome-deficient Pex3 knockout HeLa cells successfully restored peroxisome formation within 24 hours of biotin treatment. Contrarily, the same assay in Pex3/March5 double knockout HeLa failed to rescue the phenotype, as consistent with previous reports.

For the ER part, we first tested the dependence of the ER cargo, Pex16, on conventional COPII machinery for budding out from the ER. Overexpressing both the GTP-locked and GDP-locked form of Sar1, Sar1(H79G)-EGFP and Sar1(T39N)-EGFP, respectively, did not impede Pex16 localization in peroxisomes. Generation of a ER-retaining RUSH did not capture Pex16 in the ER.

In summary, my study demonstrated that the M-RUSH is capable of manipulating peroxisome de novo biogenesis in Pex3 KO HeLa cells, while Pex16 might be budded out from the ER in a COPII-independent mechanism or getting directly imported to peroxisomal membranes during steady state.

Date of Award	2025
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Yusong GUO (Supervisor)