NOA1, a Novel ClpXP Substrate, Takes an Unexpected Nuclear Detour Prior to Mitochondrial Import

Abstract

The mitochondrial matrix GTPase NOA1 is a nuclear encoded protein, essential for mitochondrial protein synthesis, oxidative phosphorylation and ATP production. Here, we demonstrate that newly translated NOA1 protein is imported into the nucleus, where it localizes to the nucleolus and interacts with UBF1 before nuclear export and import into mitochondria. Mutation of the nuclear localization signal (NLS) prevented both nuclear and mitochondrial import while deletion of the N-terminal mitochondrial targeting sequence (MTS) or the C-terminal RNA binding domain of NOA1 impaired mitochondrial import. Absence of the MTS resulted in accumulation of NOA1 in the nucleus and increased caspase-dependent apoptosis. We also found that export of NOA1 from the nucleus requires a leptomycin-B sensitive, Crm1-dependent nuclear export signal (NES). Finally, we show that NOA1 is a new substrate of the mitochondrial matrix protease complex ClpXP. Our results uncovered an unexpected, mandatory detour of NOA1 through the nucleolus before uptake into mitochondria. We propose that nucleo-mitochondrial translocation of proteins is more widespread than previously anticipated providing additional means to control protein bioavailability as well as cellular communication between both compartments.

ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells

“…We demonstrated that simultaneous overexpression of ClpX and NOA1 led to the complete degradation of NOA1 without modulating ClpP protein levels [29]. This supported the idea that ClpX was the limiting factor in determining ClpXP efficiency in muscle cells [29].…”

“…Furthermore, we confirm the involvement of a retrograde transcriptional regulation pathway mediated by the UPR mt transcription factor CHOP. In conclusion, we show that the main features of the C. elegans [9][10][11][12][13]29,33] UPR mt are conserved in mammalian cells and present ClpX overexpression as model for the mammalian UPR mt . To knockdown ClpP in cell culture, we cloned the following shRNA target sequences into the U6+2tetO [34] plasmid: ClpP sh1 5 '-GAA GCA CCT TCC ATT ACT TCT-3' and ClpP sh2 5'-GCC TCC TTC ACC TTG ACA AAC-3'.…”

“…reported a ClpXP substrate in mammalian cells, which is the mitochondrial matrix GTPase NOA1 [29,41]. We demonstrated that simultaneous overexpression of ClpX and NOA1 led to the complete degradation of NOA1 without modulating ClpP protein levels [29].…”

“…We showed that increasing ClpX protein levels alone, without changing the ClpP protein level, was sufficient to significantly increase the degradation capacity of the ClpXP complex in vivo [29].…”

“…We recently reported a mammalian substrate of ClpXP, the mitochondrial matrix GTPase NOA1 [29][30][31][32]. We showed that increasing ClpX protein levels alone, without changing the ClpP protein level, was sufficient to significantly increase the degradation capacity of the ClpXP complex in vivo [29].…”