KLHL23 and RhoGDI coordinate CDC42 inactivation ensuring membrane homeostasis.

F-Actin cytoskeleton remodeling is vital for cell migration, organ development and immune responses. The small GTPase CDC42, a key regulator of F-actin dynamics, cycles between inactive GDP- and active GTP-bound states. However, mechanisms governing CDC42 turnover and their biological significance remain unclear. Here we show that KLHL23-mediated polyubiquitylation of CDC42•GTP and RhoGDI-mediated sequestration of CDC42•GDP spatiotemporally co-inactivate CDC42, preserving membrane dynamics and homeostasis during migration. KLHL23-Cul3 acts as the E3 ligase for CDC42 degradation, with KLHL23 and RhoGDI competing for CDC42's switch II region, enhancing selectivity toward CDC42•GTP and CDC42•GDP, respectively. KLHL23 depletion disrupts membrane homeostasis, inducing excessive protrusions and promoting metastasis. Notably, the CDC42-Y64C germline variant in Takenouchi-Kosaki Syndrome escapes KLHL23-mediated degradation. Fluorescence resonance energy transfer assays reveal that KLHL23 and RhoGDI coordinately inactivate CDC42 in a spatiotemporal manner. These findings highlight the biological and clinical relevance of the KLHL23/RhoGDI-CDC42 axis, presenting new avenues for therapeutic exploration.