Intracellular protein degradation by the ubiquitin-proteasome system is ATP-dependent and the optimal ATP concentration to activate proteasome function in vitro is ~100 μM. Intracellular ATP levels are generally in the low millimolar range but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50-100 μM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated, the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly up-regulate proteasome activity upon ATP reduction under stress conditions.

Summary P0 protein of some polerovirus members can target ARGONAUTE 1 ( AGO 1) to suppress RNA silencing. Although P0 harbors an F‐box‐like motif reported to be essential for interaction with S phase kinase‐associated protein 1 ( SKP 1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO 1 degradation. Therefore, the role of P0– SKP 1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0 Br ). We found that several residues within P0 Br are required for local and systemic silencing suppression activities. Remarkably, the F‐box‐like motif mutant of P0 Br , which failed to interact with SKP 1, is destabilized in vivo . Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP 1 ortholog leads to the destabilization of P0 Br . Genetic analyses indicated that the P0 Br – SKP 1 interaction is not directly required for silencing suppression activity of P0 Br , but it facilitates stability of P0 Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of Br YV requires P0 Br . Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

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Splicing of plant organellar group II introns is under accurate nuclear control that employs many nucleus-encoded protein cofactors from various families. For mitochondrial introns, only a few splicing factors have been characterized because disruption of their functions often causes embryo lethality. Here, we report the function of Empty Pericarp8 (Emp8) in the splicing of three group II introns in mitochondria, complex I biogenesis, and seed development in maize. Emp8 encodes a P subfamily pentatricopeptide repeat protein that localizes in mitochondria. The loss-of-function mutants of Emp8 are embryo lethal, showing severely arrested embryo and endosperm development in maize. The respiration rate in the emp8 mutants is reduced with substantially enhanced expression of alternative oxidases. Transcript analysis indicated that the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 are abolished, and the cis-splicing of nad2 intron 1 is severely impaired in the emp8 mutants. These defects consequently lead to the disassembly of mitochondrial complex I and a dramatic reduction in its activity. Together, these results suggest that Emp8 is required for the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 and nad2 intron 1, which is essential to mitochondrial complex I assembly and hence to embryogenesis and endosperm development in maize.

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