Protein Import into Peroxisomes: The Principles and Methods of Studying

Abstract Peroxisomes are essential intracellular organelles that involve many metabolic processes, such as β‐oxidation of very long‐chain fatty acids and synthesis of plasmalogen and bile acids as well as generation and degradation of hydrogen peroxide. These peroxisomal functions are fulfilled by strictly and spatiotemporally regulated compartmentation of the proteins catalysing these reactions. Defects in peroxisomal protein import results in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesised on free ribosomes but transported into peroxisomes by distinct pathways determined by specific targeting signals and their receptors. The mechanism by which this is achieved has been clarified by identification of many PEX genes and the products named peroxins, the essential factors for peroxisome biogenesis. This article introduces several basic methods to investigate protein import into peroxisomes. Key Concepts Peroxisome plays an essential role in various metabolic pathways. Deficiency of peroxisomes in human causes severe foetal genetic diseases, peroxisome‐deficient disorders. Functions and the integrity of peroxisomes are archived by proper import of both matrix and membrane proteins into peroxisomes. Peroxisomal proteins are encoded by nuclear DNA, synthesised in cytosolic free ribosomes and post‐translationally imported into pre‐existing peroxisomes. Peroxisomal matrix and membrane proteins are translocated into peroxisomes via different pathways in a manner dependent on their distinct targeting signals. Many of peroxins encoded by PEX genes are responsible factors for peroxisome biogenesis and are involved in the import of peroxisomal proteins. Various experimental approaches have elucidated the molecular mechanisms underlying peroxisome biogenesis. Post‐translational import of peroxisomal proteins into isolated peroxisomes can be reproduced in vitro . Semi‐permeabilised cells, in which only the plasma membrane is selectively permeabilised, are used to investigate peroxisomal protein import, as with living cells.