Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key ...determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.
UDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT ...double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name “Parodi limit” the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70–80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a “one-size-fits-all adjustable spanner” UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.
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•UGGT MD simulations widen the conformational range observed in crystal structures•The UGGT TRXL2 domain is essential for enzymatic activity•A misfold site is closer than the Parodi limit to a glycan in UGGT clients in vitro•N-Glycan distributions suggest evolution optimizes glycoprotein surface coverage
Modenutti et al. carry out molecular dynamics simulations of UGGT, the enzyme surveying correct folding of glycoproteins, and propose a “one-size-fits all adjustable spanner” UGGT:substrate recognition model. The UGGT TRXL2 domain is essential for its function. The size of UGGT likely dictates restraints on the evolution of N-linked glycosylation sites.
SUMMARY
Directional movements impact the ability of plants to respond and adjust their growth accordingly to the prevailing light environment. The plasma‐membrane associated protein, ROOT ...PHOTOTROPISM 2 (RPT2) is a key signalling component involved in chloroplast accumulation movement, leaf positioning, and phototropism, all of which are regulated redundantly by the ultraviolet/blue light‐activated AGC kinases phototropin 1 and 2 (phot1 and phot2). We recently demonstrated that members of the NON‐PHOTOTROPIC HYPOCOTYL 3 (NPH3)/RPT2‐like (NRL) family in Arabidopsis thaliana, including RPT2, are directly phosphorylated by phot1. However, whether RPT2 is a substrate for phot2, and the biological significance of phot phosphorylation of RPT2 remains to be determined. Here, we show that RPT2 is phosphorylated by both phot1 and phot2 at a conserved serine residue (S591) within the C‐terminal region of the protein. Blue light triggered the association of 14‐3‐3 proteins with RPT2 consistent with S591 acting as a 14‐3‐3 binding site. Mutation of S591 had no effect on the plasma membrane localization of RPT2 but reduced its functionality for leaf positioning and phototropism. Moreover, our findings indicate that S591 phosphorylation within the C‐terminus of RPT2 is required for chloroplast accumulation movement to low level blue light. Taken together, these findings further highlight the importance of the C‐terminal region of NRL proteins and how its phosphorylation contributes to phot receptor signalling in plants.
Significance Statement
ROOT PHOTOTROPISM 2 (RPT2) is a key signalling component involved in chloroplast accumulation movement, leaf positioning, and phototropism, which are regulated by phototropin receptor kinases (phot1 and phot2). Here we demonstrate that RPT2 is phosphorylated by phot1 and phot2 at S591 within the C‐terminus and phosphorylation of this residue plays a role in establishing phototropism, leaf positioning and is functionally required for RPT2 to mediate chloroplast movement to low levels of blue light.
Significance Statement
This study indicates that CIPK23 is a phototropin‐interacting protein kinase that promotes blue light‐ and phototropin‐dependent stomatal opening in Arabidopsis thaliana. We ...propose that CIPK23 does not mediate plasma membrane H+‐ATPase activation but mediates inward‐rectifying K+ channel activation in stomatal opening.
SUMMARY
Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL‐interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull‐down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin‐mediated responses. We further found that blue light activation of inward‐rectifying K+ (K+in) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+‐ATPase was not. The blue light activation of K+in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+‐ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.
Protein kinases (PKs) control many aspects of plant physiology by regulating signaling networks through protein phosphorylation. Phototropins (phots) are plasma membrane–associated serine/threonine ...PKs that control a range of physiological processes that collectively serve to optimize photosynthetic efficiency in plants. These include phototropism, leaf positioning and flattening, chloroplast movement, and stomatal opening. Despite their identification over two decades ago, only a handful of substrates have been identified for these PKs. Progress in this area has been hampered by the lack of a convenient means to confirm the identity of potential substrate candidates. Here we demonstrate that the kinase domain of Arabidopsis phot1 and phot2 can be successfully engineered to accommodate non-natural ATP analogues by substituting the bulky gatekeeper residue threonine for glycine. This approach circumvents the need for radioactivity to track phot kinase activity and follow light-induced receptor autophosphorylation in vitro by incorporating thiophosphate from N6-benzyl-ATPγS. Consequently, thiophosphorylation of phot substrate candidates can be readily monitored when added or co-expressed with phots in vitro. Furthermore, gatekeeper-modified phot1 retained its functionality and its ability to accommodate N6-benzyl-ATPγS as a phosphodonor when expressed in Arabidopsis. We therefore anticipate that this chemical genetic approach will provide new opportunities for labeling and identifying substrates for phots and other related AGC kinases under in vitro and near-native in vivo conditions.
Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase ...(UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.
Summary Background Coronary stents improve immediate and late results of balloon angioplasty by tacking up dissections and preventing wall recoil. These goals are achieved within weeks after ...angioplasty, but with current technology stents permanently remain in the artery, with many limitations including the need for long-term antiplatelet treatment to avoid thrombosis. We report a prospective multicentre clinical trial of coronary implantations of absorbable magnesium stents. Methods We enrolled 63 patients (44 men; mean age 61·3 SD 9·5 years) in eight centres with single de novo lesions in a native coronary artery in a multicentre, non-randomised prospective study. Follow-up included coronary angiography and intravascular ultrasound at 4 months and clinical assessment at 6 months and 12 months. The primary endpoint was cardiac death, non-fatal myocardial infarction, or clinically driven target lesion revascularisation at 4 months Findings 71 stents, 10–15 mm in length and 3·0–3·5 mm in diameter, were successfully implanted after pre-dilatation in 63 patients. Diameter stenosis was reduced from 61·5 (SD 13·1%) to 12·6 (5·6%) with an acute gain of 1·41 mm (0·46 mm) and in-stent late loss of 1·08 mm (0·49 mm). The ischaemia-driven target lesion revascularisation rate was 23·8% after 4 months, and the overall target lesion revascularisation rate was 45% after 1 year. No myocardial infarction, subacute or late thrombosis, or death occurred. Angiography at 4 months showed an increased diameter stenosis of 48·4 (17·0%). After serial intravascular ultrasound examinations, only small remnants of the original struts were visible, well embedded into the intima. Neointimal growth and negative remodelling were the main operating mechanisms of restenosis. Interpretation This study shows that biodegradable magnesium stents can achieve an immediate angiographic result similar to the result of other metal stents and can be safely degraded after 4 months. Modifications of stent characteristics with prolonged degradation and drug elution are currently in development.
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