A spatiotemporal model of peroxisome biogenesis and Pex3 function. Peroxisome proliferation occurs by two mechanisms: division of existing peroxisomes through fission and de novo formation from the ER. Peroxisomes are formed de novo from the ER through budding and then fusion of two classes of PPV (although there may be more). This mechanism separates RING finger and docking components of the import complex into different vesicles, which are not import competent until after fusion and assembly of a complete and functional import complex. Once mature, these peroxisomes can multiply by growth and incorporate proteins and membranes from the ER. These two arms of the biogenic cascade are mediated by Pex3, which has different functions depending on its location. (1) Pex3 facilitates PMP insertion into membranes, either with assistance of a membrane translocon, or, for a subset of membrane proteins, direct insertion into the membrane. (2) Pex3 chaperones PMPs, protecting them from quality control systems such as ER-associated protein degradation (ERAD), and assisting in their lateral sorting through the ER to sites of PPV formation. (3) Pex3 and Pex19 define sites of PPV budding and recruit the scission machinery, ESCRT-III, to release them from the ER. (4) Additional functions of Pex3, such as peroxisomal tethering to the ER, are mediated by its binding proteins, such as Inp1.