Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1–4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) possesses E3 ligase activity and promotes degradation of key factors involved in the light regulation of plant development. The finding that CULLIN4 (CUL4)-Damaged DNA Binding Protein1 (DDB1) interacts with DDB1 binding WD40 (DWD) proteins to act as E3 ligases implied that CUL4-DDB1 may associate with COP1-SUPPRESSOR OF PHYA (SPA) protein complexes, since COP1 and SPAs are DWD proteins. Here, we demonstrate that CUL4-DDB1 physically associates with COP1-SPA complexes in vitro and in vivo, likely via direct interaction of DDB1 with COP1 and SPAs. The interactions between DDB1 and COP1, SPA1, and SPA3 were disrupted by mutations in the WDXR motifs of MBP-COP1, His-SPA1, and His-SPA3. CUL4 cosuppression mutants enhanced weak cop1 photomorphogenesis and flowered early under short days. Early flowering of short day-grown cul4 mutants correlated with increased FLOWERING LOCUS T transcript levels, whereas CONSTANS transcript levels were not altered. De-etiolated1 and COP1 can bind DDB1 and may work with CUL4-DDB1 in distinct complexes, but they mediate photomorphogenesis in concert. Thus, a series of CUL4-DDB1-COP1-SPA E3 ligase complexes may mediate the repression of photomorphogenesis and, possibly, of flowering time.

The evolutionarily conserved CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) is a RING and WD40 protein that functions as a substrate receptor of CULLIN4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1)-based E3 ubiquitin ligases in both plants and animals. In Arabidopsis, COP1 is a central repressor of photomorphogenesis in the form of COP1-SUPPRESSOR OF PHYA (SPA) complex(es). CUL4-DDB1-COP1-SPA suppresses the photomorphogenic program by targeting the transcription factor ELONGATED HYPOCOTYL 5 for degradation. Intriguingly, under photomorphogenic UV-B light, COP1 reverses its repressive role and promotes photomorphogenesis. However, the mechanism by which COP1 is functionally switched is still obscure. Here, we demonstrate that UV-B triggers the physical and functional disassociation of the COP1-SPA core complex(es) from CUL4-DDB1 and the formation of a unique complex(es) containing the UV-B receptor UV RESISTANCE LOCUS 8 (UVR8). The establishment of this UV-B-dependent COP1 complex(es) is associated with its positive modulation of ELONGATED HYPOCOTYL 5 stability and activity, which sheds light on the mechanism of COP1's promotive action in UV-B-induced photomorphogenesis.light signaling | protein complex | posttranscriptional regulation I n response to light and darkness, plant seedlings establish lightgrown and dark-grown phenotypes via a series of developmental changes, termed photomorphogenesis and skotomorphogenesis, respectively. CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is a known RING E3 ubiquitin ligase that has been evolutionally conserved from plants to humans (1, 2). It was originally identified by genetic screens for seedlings of Arabidopsis thaliana that exhibit constitutive photomorphogenesis in darkness (1, 3), as a key member of the pleiotropic CONSTITUTIVE PHOTOMOR-PHOGENIC/DE-ETIOLATED/FUSCA (COP/DET/FUS) gene family. These COP/DET/FUS proteins biochemically contribute to three entities: the COP1-SUPRESSOR OF PHYA (SPA) complex(es), the COP9 signalosome (CSN), and the COP10-DET1-Damaged DNA Binding Protein 1 (DDB1) (CDD) complex. COP1-SPA, independent of CDD but in concert with CULLIN4-DDB1 (CUL4-DDB1), targets photomorphogenesis promoting transcription factors including ELONGATED HYPOCOTYL 5 (HY5) for the ubiquitin-proteasome system-mediated degradation, so as to repress the traditional photomorphogenesis triggered by far-red and visible light (4, 5).Intriguingly, in contrast to their antagonistic roles in the traditional photomorphogenesis, COP1 and HY5 both take positive parts in low-fluence and long-wavelength UV-B-induced photomorphogenesis. This response is initiated by the UV-B receptor UV RESISTANCE LOCUS 8 (UVR8) which absorbs UV-B through its internal chromophore tryptophan residues (6, 7). UVR8 then monomerizes to interact with the UV-B-inducible protein COP1 for downstream signaling (8-10). The physical manifestations of this process include hypocotyl shortening, anthocyanin accumulation, and tolerance against damaging UV-B. The loss of either COP1 or HY5 has previously been shown to result in de...