Peroxisomes are a new addition to the secretory pathway and form the endpoint of a new ER exit route. Although membrane proteins and lipids are acquired from the ER, peroxisomes handle the import of matrix proteins from the cytosol themselves via a peroxisome-specific translocon. We provide a new framework for organelle biogenesis whereby different ER-derived vesicles fuse to form a peroxisomal pre-compartment. This obligatory step in peroxisome biogenesis is necessary to both temporally and spatially restrict the assembly of the peroxisomal translocon, so that it is active only in the peroxisomal membrane. The vesicles that bud from the ER act as two-component ‘glue’, each carrying a ‘half’ peroxisomal translocon complex. Upon heterotypic vesicle fusion the two half-translocons now share one compartment allowing formation of active peroxisomal translocon complexes and uptake of peroxisomal enzymes. Our findings demonstrate a remarkable mechanism of how the biochemical identity of organelles (ER) is maintained.
