Peroxisomes are found in virtually all eukaryotic cells, where they play varied but essential metabolic roles, exemplified by the catastrophic effects of mutations that compromise peroxisome biogenesis and function. This review will aim to provide an accessible introduction to peroxisome biogenesis and protein import for the non-specialist, and draws together recent advances in peroxisome protein targeting and import in plants, animals and yeasts, seeking to define common themes and highlight variations. Despite much progress, many aspects of peroxisome biology remain an enigma, and current questions and controversies in the field are highlighted and discussed.
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