An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.     

Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.     

GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi‐enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non‐exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter‐mediated pathway and the vesicle trafficking‐mediated pathway. No molecules involved in the vesicle trafficking‐mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan‐colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis‐sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome‐like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9‐mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.     

A rich and bountiful harvest: Key discoveries in plant cell biology

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke’s discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

Seven senior plant cell biologists reflect on foundational contributions to a variety of topics, including pollen tube signaling, cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology.     

Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex

Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.     

Immunohistochemical colocalization of glucagon,serotonin, and aromatic L-amino acid decarboxylase in islet A cells of chicken pancreas

Summary The identity of monoamine-emitted, formaldehyde-induced fluorescence in some pancreatic islet cells was studied in pancreatic tissue of male chickens by fluorescence and immunohistochemistry either on the same tissue section or on serial tissue sections. Pancreatic islet cells emitting intense formaldehyde-induced fluorescence also react immunohistochemically with antisera directed against glucagon, serotonin and aromatic L-amino acid decarboxylase. These results show that chicken pancreatic islet A cells contain glucagon, serotonin, and aromatic L-amino acid decarboxylase, an enzyme involved in the synthesis of serotonin. The islet B cells identified with anti-insulin immunoreactivity, which displayed a very weak formaldehyde-induced fluorescence, did not react with anti-serotonin serum.     

Analysis of the kinetics of CO binding to neuronal nitric oxide synthase by flash photolysis: dual effects of substrates, inhibitors, and tetrahydrobiopterin

The effects of substrates, inhibitors and tetrahydrobiopterin (H4B) on CO rebinding to the isolated heme-bound oxygenase domain (nNOSox) of neuronal nitric oxide synthase were examined by laser flash photolysis. The rate constant of CO recombination with substrate and inhibitor-free nNOSox in the absence of H4B was 1.0 x 10(6) M(-1) s(-1). The addition of H4B led to a marked decrease in the rate to 0.59 x 10(6) M(-1) s(-1). Interestingly, the substrates, L-Arg and N-hydroxy-L-Arg (NHA), altered CO binding behavior in that the binding rate was modified to CO concentration-independent, both with and without H4B. In the absence of H4B, agmatine, NG-monomethyl-L-Arg (NMMA) and NG-nitro-L-Arg methyl ester (NAME) decreased the CO concentration-dependent rate constants of rebinding by half (0.43 x 10(6) M(-1) s(-1) for the NMMA-bound complex), whereas N6-(l-iminoethyl)-L-Lys (NIL) and 7-nitro-1H-indazole (7-NI) increased the rate constants by more than 70% (up to 2.1 x 10(6) M(-1) s(-1) for the NIL-bound complex). In the presence of H4B, the binding rate was independent of CO concentration for the agmatine-bound complex. The differential effects of the inhibitors on the CO concentration-dependent rate constants were significantly diminished for the H4B-bound system. Interestingly, these variable effects of inhibitors on the CO binding rate were more pronounced in the absence of H4B. Accordingly, we suggest that H4B significantly influences CO binding by altering the CO access channel, and further reduces the divergent effects of different inhibitors.     

Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for beta-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From approximately 3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.     

Blue light-induced association of phototropin 2 with the Golgi apparatus

Phototropins 1 and 2 (phot1 and phot2) function as blue light (BL) photoreceptors for phototropism, chloroplast relocation, stomatal opening and leaf flattening in Arabidopsis thaliana. Phototropin consists of two functional domains, the N-terminal photosensory domain and the C-terminal Ser/Thr kinase domain. However, little is known about the signal transduction pathway that links the photoreceptors and the physiological responses downstream of BL perception. To understand the mechanisms by which phot2 initiates these responses, we transformed the phot1phot2 double mutant of Arabidopsis with constructs encoding translationally fused phot2:green fluorescent protein (P2G). P2G was fully functional for the phot2-specific physiological responses in these transgenic plants. It localized strongly to the plasma membrane and weakly to the cytoplasm in the dark. Upon illumination with BL, punctate P2G staining was formed within a few minutes in addition to the constitutive plasma membrane staining. This punctate distribution pattern matched well with that of the Golgi-localized KAM1DeltaC:mRFP. Brefeldin A (BFA), an inhibitor of vesicle trafficking, induced accumulation of P2G around the perinuclear region even in darkness, but the punctate pattern was not observed. After treatment of these cells with BL, P2G exhibited the punctate distribution pattern that matched with that of the Golgi marker. Hence, the light-dependent association of P2G with the Golgi apparatus was BFA-insensitive. A structure/function analysis indicated that the kinase domain was essential for the Golgi localization of phot2. The BL-induced Golgi localization of phot2 may be one of important signaling steps in the phot2 signal transduction pathway.