Updated project metadata. MDVs are implicated in diverse physiological processes e.g. mitochondrial quality control and are linked to various neurodegenerative diseases. However, their specific cargo composition and complex molecular biogenesis is still unknown. Here we report for the first time the proteome and lipidome of steady-state TOMM20+-MDVs. We identified 106 novel high confidence MDV cargoes and verified several candidates by super-resolution microscopy including 5 members of the TOM import complex. Additionally, we demonstrate that MDVs deliver fully assembled protein complexes like the TOM complex for lysosomal degradation, thus filling a crucial mitochondrial quality control niche. Moreover, we show key biogenesis steps of MDVs starting with the MIRO1/2-dependent formation of thin membrane protrusions pulled along microtubule filaments, followed by MID49/MID51/MFF-dependent recruitment of the dynamin family GTPase DRP1 and finally DRP1-dependent scission. We conclude that catalysing scission during MDV biogenesis demonstrates a novel function of DRP1, distinct from its role in mitochondrial division.