Identification of Calcium-Sensing Receptor (CAS) as a Phytaspase Interactor: Implications for the CAS Trafficking...

Proteolytic enzymes play important and diverse roles in regulating protein homeostasis in living cells. Phytaspase, a member of the plant subtilisin-like protease family possessing a 'caspase-like' selectivity and specificity of hydrolysis of its targets, is a cell death-related protease. Phytaspase displays a peculiar trafficking pattern and is involved in the execution of plant cell death induced by biotic and abiotic stresses. To elucidate the molecular mechanisms of phytaspase functioning, a search for Nicotiana tabacum phytaspase (NtPhyt)-interacting proteins was performed using the BioID approach. This search identified the calcium-sensing receptor (CAS) protein as a candidate NtPhyt interactor. Direct interaction of NtPhyt with N. tabacum CAS (NtCAS) was corroborated by an in vitro analysis utilizing the purified proteins. Furthermore, NtPhyt was shown to introduce five cleavages in the CAS molecule in vitro. For NtCAS-EGFP produced in N. benthamiana leaves, dual localization to chloroplasts and the cell periphery, including the plasma membrane, was observed, whereas NtPhyt proteolytic activity was undetectable in the chloroplasts. A CAS precursor protein apparently preserving the chloroplast transit peptide was found to accumulate upon a light-to-dark transition and to disappear in the presence of elevated levels of NtPhyt. A requirement for CAS for stress-induced retrograde transport of NtPhyt was uncovered, and partial co-localization of CAS with NtPhyt was observed in cells subjected to oxidative stress. Our results are consistent with the model according to which CAS transiently localizes to the plasma membrane as an intermediate location on its way to its final destination, the chloroplasts, and interacts with NtPhyt at this stage.