The peroxisome is an organelle conserved throughout eukaryotes, ranging from single-celled yeast to multi-cellular organisms including flies and humans. Peroxisome biogenesis involves formation of the peroxisomal membrane followed by import of luminal matrix proteins. Peroxisomes can be generated de novo from the endoplasmic reticulum (ER) or by fission of pre-existing peroxisomes. Proteins controlling peroxisome formation are collectively termed peroxins, and over 30 peroxins have now been identified. Peroxisomes depend on a diverse group of mechanisms to ensure the correct targeting of matrix and membrane proteins. However, the membrane biogenesis pathway is poorly characterised and currently limited to only three factors: Pex3, Pex16 and Pex19. Peroxisomal membrane proteins (PMPs) are targeted to peroxisomes by two distinct pathways; directly to peroxisomes or indirectly via the ER. Mutant peroxins are the cause of peroxisome biogenesis disorders in humans so it is essential to characterise their individual molecular mechanisms. Limited peroxisome research has been conducted using Drosophila melanogaster as a model system, but current studies indicate peroxins and cellular pathways are evolutionary conserved. Previously, our lab performed a genome-wide screen in Drosophila S2R+ cells to identify candidate peroxins. No mitochondrial derivative (NMD) was the top hit based on its knockdown phenotype of an import defect of PTS1 proteins. The AAA+ ATPase NMD is a conserved integral membrane protein that dually localises to peroxisomes and mitochondria. Loss of NMD, via RNAi or CRISPR, led to a lack of PMP import and mislocalisation of Pex3. NMD contains multiple sorting signals, displaying distribution between ER, peroxisomes and mitochondria. NMD was shown to interact directly with Pex16, a well-known peroxisomal membrane biogenesis factor, and that interaction is dependent on ATP binding. Further coimmunoprecipitation studies identified that NMD interacts with multiple peroxins as well as the ER translocon Sec61a. Overexpression of a dominant negative mutant of NMD defective in ATP hydrolysis, led to the mislocalisation of Pex3 and an import defect of PTS1 proteins, demonstrating ATPase activity is essential for function. Homologues of NMD exist in humans (ATAD1) and yeast (Msp1); but function in membrane assembly is not conserved. Analysis of the function of Msp1 revealed a genetic interaction with the yeast peroxin Pex25 in controlling peroxisome numbers. Collectively, the results suggest NMD is a novel component of the PMP import machinery in Drosophila, but that its role is not conserved in other model organisms.