Human Peroxin PEX3 Is Co‐translationally Integrated into the ER and Exits the ER in Budding Vesicles

The long‐standing paradigm that all peroxisomal proteins are imported post‐translationally into pre‐existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) inside the endoplasmic reticulum (ER). In mammals, the mechanisms of ER entry and exit of PMPs are completely unknown. We show that the human PMP PEX3 inserts co‐translationally into the mammalian ER via the Sec61 translocon. Photocrosslinking and fluorescence spectroscopy studies demonstrate that the N‐terminal transmembrane segment (TMS) of ribosome‐bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3‐containing ribosome•nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins Sec61α and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP‐dependent process. This study identifies early steps in human peroxisomal biogenesis by demonstrating sequential stages of PMP passage through the mammalian ER. Some peroxisomal membrane proteins (PMPs) are targeted first to the endoplasmic reticulum (ER) prior to being transported to peroxisomes. The mechanisms that mediate ER‐passage of PMPs in mammalians are unknown. We demonstrate that the human PMP PEX3 inserts co‐translationally into the ER by an SRP/Sec61‐translocon‐dependent multistep pathway. Subsequently, PEX3 exits the ER via budding vesicles in an energy‐consuming reaction. Hence, we demonstrate how the ER is seeded with PMPs, and highlight the significance of ER‐to‐peroxisome vesicle trafficking in human organelle biogenesis.