The Cytokinin Oxidase/Dehydrogenase CKX1 Is a Membrane-Bound Protein Requiring Homooligomerization in the Endoplasmic Reticulum for Its Cellular Activity

Abstract

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 localizes predominantly to the endoplasmic reticulum (ER) in Arabidopsis (). 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 amino-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 strong support for the cytokinin signaling from the ER and for the functional relevance of the cytokinin pool in this compartment.

Evolution and roles of cytokinin genes in angiosperms 2: Do ancient CKXs play housekeeping roles while non-ancient CKXs play regulatory roles?

“…In addition, ancient and non-ancient CKXs present differential subcellular localizations (Table 1). Studies on AtCKXs have revealed that the ancient AtCKX7 uniquely localizes to the cytosol, whereas the non-ancient AtCKXs predominantly localize to the endoplasmic reticulum (ER), vacuole or apoplast 11,13,40 . This discrepancy in subcellular localizations between ancient and non-ancient CKXs is likely related to their respective housekeeping and regulatory roles in angiosperms.…”

Root Engineering in Barley: Increasing Cytokinin Degradation Produces a Larger Root System, Mineral Enrichment in the Shoot and Improved Drought Tolerance

“…The two CKX gene products, CKX1 and CKX2, differ in their subcellular localization and biochemical characteristics. A tagged CKX1 protein was shown to localize predominantly to the endoplasmic reticulum (Niemann et al, 2018), while a CKX2-GFP fusion protein localized to the endoplasmic reticulum and possibly also to the apoplastic space Zürcher et al, 2016). Furthermore, the CKX1 protein preferred CK ribosides and N 9 -glucosides as substrates, while N 6 -isopentenyladenine (iP) and iP riboside were preferred by CKX2 (Galuszka et al, 2007).…”

Auxins and Cytokinins—The Role of Subcellular Organization on Homeostasis

“…It seems that the main site of CK inactivation is localized in the apoplast by CKX2 and CKX4-6 [ 151 ]. CKX1 and 3 were initially predicted as mitochondrial based on in silico experiments [ 152 ], although later GFP fusions showed that these two enzymes are predominantly targeted to vacuole, with some observed signal also in ER [ 151 ] where they are catalytically active [ 153 ]. In the case of CKX7, the lack of a signal peptide suggests that it is localized to the cytosol [ 154 ] ( Figure 3 ).…”