b -Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Speciﬁcities; Physical Interaction with Nitrate Reductase Speciﬁcally Occurs via AKIN b 1-Subunit 1[C][OA]

The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one a -type catalytic subunit and two g - and b -type regulatory subunits. In yeast it has been proposed that the b -type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three b -type subunits of Arabidopsis ( Arabidopsis thaliana ; AKIN b 1, AKIN b 2, and AKIN b 3) restore the growth phenotype of the yeast sip1 D sip2 D gal83 D triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKIN b promoter:: b -glucuronidase transgenic lines, reveal different and speciﬁc patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the b -type subunits are involved in the speciﬁcity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each b -type subunit in plants. of 2,454 bp of the AKIN b 3 promoter and the 5 # untranslated region (UTR; containing the two leader introns) were ampliﬁed by PCR using 5 # -GATGCCCACCACCAACCAA-3 # and 5 # -GATTTTGACTGTTC-ATCTTGG-3 # oligonucleotides modiﬁed with Not I restriction sites. For AKIN b 1::GUS and AKIN b 2::GUS constructs, PCR experiments were performed using, respectively, 5 # -TGTGGAAGATTTAGTGTC-3 # and 5 # -GTCTACCCTTTACGCTTGCCG-3 # oligonucleotides and 5 # -CTCAGCGT-CTTCGTCTTCAAC-3 # and 5 # -TCAACCACCAGTCTTCCC-3 # oligonucleo- tides on AKIN b 1 and AKIN b 2 gene promoters and 5 # UTR. Products, 800 bp for AKIN b 1 and 1,200 bp for AKIN b 2 were fused in frame to the 5 # end of the UIDA gene:: NOS terminator insert (Jefferson et al., 1987) by insertion between the Bam HI and Hin dIII sites of the pGUS vector to yield the AKIN b 1 ::GUS and AKIN b 2 ::GUS constructs that were then subcloned into the pPF111 binary vector between Hin dIII and Eco RI sites. two independent experiments. The vectors described above were used to produce 35 S -Met-labeled proteins by in vitro transcription/translation using a TNTT7 quick transcription/ translation lysate system (Promega). Six milligrams of ProteinA-Sepharose (Sigma P3391) was incubated withor without 5 m L of labeled NR2in 400 m L of buffer I (MOPS 25 m M pH 7.5, NaCl 50 m M , glycerol 10% [v/v], bovine serum albumin 6 mg/mL, EDTA 1 m M , Tween 20 0.5% [v/v], NaN 3 0.02% [v/v]), at 4 (cid:3) Cfor1 h.Fivemicrolitersof alabeled protein (orpeptide) was addedforone more hour. The Sepharose beads were then centrifuged (5,000 g ) and washed eight times with 1 mL of the buffer I. Proteins were then detached from the ProteinA-Sepharose in 20 m L of Laemmli buffer at 85 (cid:3) C for 5 min and separated in SDS-PAGE (10%–15% [w/v] acrylamide). The gel was dried and exposed to x-ray ﬁlm overnight at room temperature. Interaction was con- sidered positive by comparing the binding of the partner of NR2 on the ProteinA-Sepharose, in the presence or absence of NR2. Each in vitro inter- action assay has been performed at least three times.