Cytohesin proteins are guanine nucleotide exchange factors (GEFs) for ARF GTPases, particularly ARF1 and ARF6. Although Arf1 and Arf6 deficiency leads to embryonic lethality, the in vivo roles of cytohesins remain poorly characterized. In this study, we investigated the functions of cytohesin-2 both in vivo and in vitro. Strikingly, full knockout of cytohesin-2 in mice results in perinatal lethality within 20 h of birth. Employing mass spectrometry-based organellar proteomics for the cellular analysis of cytohesin-2 function, we discovered an altered Golgi apparatus in cytohesin-2-deficient C2 myoblasts. Specifically, immunofluorescence analysis demonstrated a significant reduction in Golgi volume compared with the control, which was restored by reintroduction of cytohesin-2 in an ARF-GEF-independent manner. Moreover, we discovered that canonical Golgi functions are impaired in cytohesin-2 deficiency: Peanut agglutinin staining showed a significant reduction in galactose/N-acetyl-galactosamine at the cell level. In addition, global protein secretion was markedly reduced in neonatal cytohesin-2 knockout mice, as determined by quantitative mass spectrometry-based proteomics. Together, our findings establish cytohesin-2 as an essential regulator of perinatal development and as a mediator of Golgi maintenance.
© 2026 Küsters et al.