The cytokinin oxidase/dehydrogenase CKX1 is a membrane-bound protein requiring homooligomerization in the endoplasmic reticulum for its cellular activity.

Degradation of the plant hormone cytokinin is controlled by cytokinin oxidase/dehydrogenase (CKX) enzymes. The molecular and cellular behavior of these proteins is still largely unknown. In this study, we show that CKX1 is a type-II single-pass membrane protein that predominantly localizes to the endoplasmic reticulum (ER). This indicates that this CKX isoform is a bona fide ER protein directly controlling the cytokinin which triggers the signaling from the ER. By using various approaches, we demonstrate that CKX1 forms homodimers and homooligomers in vivo. The N-terminal part of CKX1 was necessary and sufficient for the protein oligomerization as well as for targeting and retention in the ER. Moreover, we show that protein-protein interaction is largely facilitated by transmembrane helices and depends on a functional GxxxG-like interaction motif. Importantly, mutations rendering CKX1 monomeric interfere with its steady-state localization in the ER and cause a loss of the CKX1 biological activity by increasing its ER-associated degradation. Therefore, our study provides evidence that oligomerization is a crucial parameter regulating CKX1 biological activity and the cytokinin concentration in the ER. The work also lends a strong support for the cytokinin signaling from the ER and for the functional relevance of the cytokinin pool in this compartment.