AbstractAlthough the negative regulator of nuclear import (NRNI) BRCA1 binding protein 2 (BRAP2) is highly expressed in testis, its role is largely unknown. Here we address this question by documenting the BRAP2 interactome from human testis, using the yeast 2-hybrid system to identify BRAP2-interacting proteins with roles in diverse cellular processes, including regulation of the actin cytoskeleton, ubiquitinylation, cell cycle/apoptosis and transcription. Interaction with BRAP2 in adult mouse testis with three of these, PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), A-Kinase anchor protein (AKAP3) and DNA methyl transferase 1 (DNMT1), was confirmed by coimmunoprecipitation assays. BRAP2's ability to inhibit PHLPP1 and DNMT1 nuclear localisation was also confirmed by quantitative confocal microscopy. Importantly, the physiological relevance thereof was implied by the cytoplasmic localisation of PHLPP1, AKAP3 and DNMT1 in pachytene spermatocytes/round spermatids where BRAP2 is present at high levels and nuclear localisation of PHLPP1 and DNMT1 in spermatogonia concomitant with lower levels of BRAP2. Interestingly, BRAP2 was also present in murine spermatozoa, in part colocalised with AKAP3. Together the results indicate for the first time that BRAP2 may play an important NRNI role in germ cells of the testis, with an additional, scaffold/structural role in mature spermatozoa.