No description available.

No description available.

Understanding the mechanisms plants utilize for seed storage protein (SSP) synthesis, transport and deposition have the potential rewards of enabling high yields of modified or foreign proteins. Hayashi et al. (1999) indicated that the machinery devoted to the synthesis of protein storage vacuoles in cotyledon cells can be induced in vegetative tissue by the constitutive expression of a pumpkin 2S albumin phosphinothricin-acetyl-transferase gene fusion (pumpkin 2S-PAT) resulting in the biogenesis of precursor-accumulating (PAC) vesicles in Arabidopsis leaves. This discovery was the impetus behind the work described which sought to examine this phenomenon further by ectopically evoking SSP trafficking and vesicle biogenesis machinery in leaves. With the aim of elucidating the mechanisms necessary to evoke PAC vesicle biogenesis, a suite of constructs including the pumpkin 2S-PAT and analogous napin-PAT and napin-GFP variants were synthesized. Analysis of these transgenes in Arabidopsis revealed that the pumpkin 2S albumin has a capacity unique from napin peptides to result in fusion protein accumulation. Further, the truncated pumpkin 2S albumin peptide and the pumpkin 2S albumin C-terminus were found to direct deposition to vesicles; however, the C-terminus alone was not enough to direct deposition to vesicles unless combined with a significantly shortened napin peptide. An increased ER protein throughput was correlated to trafficking of the fusion protein by Golgi-independent mechanisms resulting in stable accumulation of the unprocessed protein whereas less ER throughput indicated passage through the Golgi-dependent pathway resulting in accumulation of a processed variant. At the level of gene expression, as examined by a microarray study, both inducible and constitutive ectopic expression of pumpkin 2S-PAT resulted in substantial perturbations of the endomembrane system affecting protein folding, flowering time and ER-associated biosynthetic functions which indicated that modulation of flowering time and photoperiodism are highly dependent on protein trafficking and vacuolar biogenesis mechanisms and that high ER protein throughput occurs at the expense of biosynthesis and cessation of ER functioning.

The activity of vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.

X-linked myopathy with excessive autophagy (XMEA) is a skeletal muscle disorder inherited in recessive fashion, affecting boys and sparing carrier females. Onset is in childhood with weakness of the proximal muscles of the lower extremities, progressing slowly to involve other muscle groups. Pathological analysis of skeletal muscle biopsies shows no inflammation, necrosis or apoptosis. Instead, forty to 80% of fibers exhibit giant autophagic vacuoles with heterogeneous degradative content. Numerous critical functions of all cells are compartmentalized in particular pH environments established by the intracellular transmembrane V-ATPase proton pump complex. Assembly of this complex, directed by the Vma21p chaperone, is well-studied in yeast but completely unknown in other organisms. The aim of my project was a better understanding of XMEA pathogenesis, with a focus on finding the disease-causing gene. In this thesis, I identify mutations in XMEA patients in a novel, previously uncharacterized gene, which we name VMA21. Most of the mutations are located in splicing-relevant positions and decrease splicing efficiency. After establishing that XMEA is caused by hypomorphic alleles of the VMA21 gene, I show that VMA21 is the diverged human orthologue of the yeast Vma21p protein, and that like Vma21p, it is an essential assembly chaperone of the V-ATPase. Decreased VMA21 reduces V-ATPase activity, resulting in altered lysosomal pH and a blockage at the degradative step of autophagy. Towards understanding disease pathogenesis, I show evidence of compensatory autophagy upregulation consecutive to the impaired clearance. Accumulated autolysosomes due to increased autophagy continue to face the degradative block and are slow to disappear. Instead, they merge to each other and form the characteristic giant XMEA vacuoles. These results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy. This work describes the clinical outcome at the cusp of tolerable reduction in V-ATPase, with implications on common diseases like osteoporosis and cancer metastasis, where increased V-ATPase activity is an important component. Our XMEA patients show that the safety margin of reducing V-ATPase activity in humans is wide, increasing the potential to utilize chemical or biological V-ATPase inhibitors as possible therapies.

X-linked myopathy with excessive autophagy (XMEA) is a skeletal muscle disorder inherited in recessive fashion, affecting boys and sparing carrier females. Onset is in childhood with weakness of the proximal muscles of the lower extremities, progressing slowly to involve other muscle groups. Pathological analysis of skeletal muscle biopsies shows no inflammation, necrosis or apoptosis. Instead, forty to 80% of fibers exhibit giant autophagic vacuoles with heterogeneous degradative content. Numerous critical functions of all cells are compartmentalized in particular pH environments established by the intracellular transmembrane V-ATPase proton pump complex. Assembly of this complex, directed by the Vma21p chaperone, is well-studied in yeast but completely unknown in other organisms. The aim of my project was a better understanding of XMEA pathogenesis, with a focus on finding the disease-causing gene. In this thesis, I identify mutations in XMEA patients in a novel, previously uncharacterized gene, which we name VMA21. Most of the mutations are located in splicing-relevant positions and decrease splicing efficiency. After establishing that XMEA is caused by hypomorphic alleles of the VMA21 gene, I show that VMA21 is the diverged human orthologue of the yeast Vma21p protein, and that like Vma21p, it is an essential assembly chaperone of the V-ATPase. Decreased VMA21 reduces V-ATPase activity, resulting in altered lysosomal pH and a blockage at the degradative step of autophagy. Towards understanding disease pathogenesis, I show evidence of compensatory autophagy upregulation consecutive to the impaired clearance. Accumulated autolysosomes due to increased autophagy continue to face the degradative block and are slow to disappear. Instead, they merge to each other and form the characteristic giant XMEA vacuoles. These results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy. This work describes the clinical outcome at the cusp of tolerable reduction in V-ATPase, with implications on common diseases like osteoporosis and cancer metastasis, where increased V-ATPase activity is an important component. Our XMEA patients show that the safety margin of reducing V-ATPase activity in humans is wide, increasing the potential to utilize chemical or biological V-ATPase inhibitors as possible therapies.

La cardosine A est une protéase aspartique identifiée il y a plus de 20 ans dans les cellules du chardon Cynara cardunculus. Sa distribution dans tous les tissus de la plante et ses caractéristiques enzymatiques ont été caractérisées par approches biochimiques. La cardosine A a des fonctions essentielles dans la reproduction, la mobilisation de réserves protéiques, et le remaniement de membranes. Pour assumer ces différentes fonctions, la cardosine A doit pouvoir transiter et s’accumuler dans différents compartiments intracellulaires : vacuole de stockage, vacuoles lytiques, ou autres compartiments membranaires. Il n’y a cependant que très peu de données disponibles sur les mécanismes de biosynthèse, de tri, de transport et d’adressage aux différents compartiments cellulaires. De récents travaux suggèrent que l’expression en modèle hétérologue pourrait être utilisée pour une meilleure compréhension de la biologie intracellulaire de la cardosine A. Les résultats de cette étude montrent que l’expression transitoire de la cardosine A dans les feuilles de Nicotiana tabacum est un bon modèle expérimental pour explorer le transport de la cardosine A dans la cellule. En effet dans ce système les mécanismes de maturation et de transport de la protéine à la vacuole sont conservés. De plus, une lignée stable d’Arabidopsis thaliana exprimant la cardosine A sous promoteur inductible s’est également avérée un bon modèle d’étude du transport intracellulaire de la cardosine A. L’utilisation de ces systèmes hétérologues a permis de combiner l’expression de formes mutées de la cardosine A (dans lesquelles des séquences spécifiques ou des acides aminés avaient été tronqués ou modifiés) avec des approches de biochimie et d’imagerie cellulaire pour identifier des signatures moléculaires responsables de l’adressage vacuolaire de la protéine. Nos résultats montrent que la cardosine A a deux déterminants vacuolaires dans sa séquence protéique : le domaine “PSI”, qui définit un déterminant d’adressage vacuolaire original et propre à certaines protéases aspartiques, et un peptide C-terminal appartenant à la classe bien définie des ctVSD. De plus, les résultats montrent que la présence de ces deux déterminants illustre la capacité d’emprunter deux routes distinctes pour atteindre la vacuole : le domaine PSI peut permettre d’attendre la vacuole sans passer par le Golgi, tandis que le domaine C-ter négocie un transport classique Reticulum, Golgi, Prévacuole, Vacuole. Cette capacité à choisir deux routes différentes n’est pas observée pour la cardosine B, autre protéase aspartique du chardon présentant une haute homologie de séquence avec la cardosine A. Pour expliquer cette capacité de la cardosine A à emprunter deux routes vacuolaires différentes, l’hypothèse d’un rôle possible de la glycosylation dans le tri des protéines entre les deux routes vacuolaires est alors étudiée. L’expression de la cardosine A dans les graines en germination d’Arabidopsis thaliana révèle que la protéine peut s’accumuler d’une manière différentielle dans les graines en absence de germination ou pendant la germination, tout au long du système endomembranaire jusqu’à la vacuole de réserve ou dans les vacuoles lytiques en formation. Les expériences de blocage de transport du Reticulum au Golgi n’ont pas permis de conclure d’une manière certaine si les accumulations vacuolaires dérivaient d’un transport pouvant court-circuiter le Golgi comme dans les feuilles de Nicotiana. Au total, la cardosine A constitue une protéine modèle pour étudier les transports vacuolaires chez Nicotiana tabacum and Arabidopsis thaliana, deux systèmes hétérologues qui permettent de développer des méthodes complémentaires pour une exploration fonctionnelle des mécanismes impliqués. Cette étude permet de contribuer à une meilleure connaissance de la biologie des cardosines en particulier, et des protéases aspartiques en général. / The aspartic proteinase cardosin A is a vacuolar enzyme found to accumulate in protein storage vacuoles and lytic vacuoles in the flowers and in protein bodies in seeds of the native plant cardoon. Cardosin A has been first isolated almost two decades ago and has been extensively characterized since, both in terms of distribution within the tissues and of enzyme biochemistry. In the native system, several roles have been addressed to cardosin A in reproduction, mobilization of reserves and membrane remodeling. To participate in such diverse events, cardosin A must accumulate and travel to different compartments inside the cell: protein storage vacuoles, lytic vacuoles, cytoplasmic membrane (and eventually outside the cell). However, not much information is available regarding cardosin A biogenesis, sorting or trafficking to the different compartments. Recent studies have approached the expression of cardosin A in Arabidopsis thaliana and Nicotiana tabacum. These preliminary observations were the starting point of a detailed study of cardosin A expression, localisation, sorting and trafficking routes, resourcing to several and very different methods. It has been showed that transient expression of cardosin A in Nicotiana tabacum leaf is a good system to explore cardosin A trafficking inside the cell, as the protein is processed in a similar manner as the control and accumulates in the vacuole. Furthermore, an Arabidopsis thaliana line expressing cardosin A under an inducible promoter was explored to understand cardosin A dynamics in terms of vacuolar accumulation during seed germination events. Similarly to the Nicotiana tabacum one, this system was also validated for cardosin A expression and it allowed to conclude that the protein’s expression did not retrieved any phenotype to the cells or individuals. However, experiments conducted in BY-2 cells revealed to be inconclusive since cardosin A expression in this system is not predictable. The data obtained along this work using several cardosin A mutated forms, lacking specific domains or point-mutated, allowed to determine that cardosin A has two Vacuolar Sorting Determinants in its protein sequence: the PSI, an unconventional sorting determinant, and the C-terminal peptide, a C-terminus sorting determinant by definition. Furthermore, it was also demonstrated that each domain represents a different route to the vacuole: the PSI bypasses the Golgi Apparatus and the C-terminal peptide follows a classic Endoplasmic Reticulum-Golgi Apparatus-Prevacuole route to the vacuole. This difference in the trafficking routes is not observed for cardosin B sorting determinants as both the PSI and C-terminal peptide from cardosin B needs to pass the Golgi Apparatus to reach the vacuole. A putative role for glycosylation in the trafficking routes is further discussed as cardosin A PSI, contrary to cardosin B, is not glycosylated. The production of mutants affecting cardosin A glycosylation sites supported this idea. Moreover, cardosin A expression in germinating Arabidopsis thaliana seeds revealed a differential accumulation in non-germinated and germinated seedlings. Cardosin A was detected along the secretory pathway to the Protein Storage Vacuole in association with the Endoplasmic Reticulum, Golgi Apparatus, Prevacuole and newly formed Lytic Vacuoles. The drug Brefeldin A caused the protein to be retained in the Golgi Apparatus, despite some amount being still detected in the vacuole, not being clear if the Golgi Apparatus bypass observed in Nicotiana tabacum leaves occurs in this system. As a whole, cardosin A confirmed to be a good model to study vacuolar sorting in these two systems that complement each other in terms of approaches available. This study provided good results in order to understand in more detail cardosin A biology in particular and vacuolar trafficking of plant Aspartic Proteinases as a group.

The vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V-0 and V-1 subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V1 components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V-1 translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.

Bone resorption involves osteoclast-mediated acidification via a vacuolar type H+-ATPase (V-ATPase) found in lysosomes and at the ruffled border membrane. V-ATPases are proton pumps that include the a3 subunit, one of four isoforms (a1-a4) in mammals. The a3 isoform is enriched in osteoclasts where it is essential for bone resorption. Over 50% of humans with osteopetrosis have mutations in the a3 subunit and a3 mutations in mouse also result in osteopetrosis. A mouse founder with an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This mouse bears a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit resulting in the replacement of a highly conserved amino acid, arginine 740, with serine (R740S). The heterozygous mice (+/R740S) exhibit high bone density but otherwise have a normal appearance, size and weight. Osteoblast parameters are unaffected whereas osteoclast number and marker expression are increased along with a decreased number of apoptotic osteoclasts. V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport along with wild type levels of ATP hydrolysis, indicating that the R740S mutation uncouples ATP hydrolysis from proton transport. The mutation however has no effect on ruffled border formation or polarization of +/R740S osteoclasts. Mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis than +/R740S mice and die by postnatal day 14. Similarly to the mouse models that lack the a3 subunit (oc/oc and Tcirg1-/-) R740S/R740S osteoclasts do not polarize and lack ruffled border membranes. However R740S/R740S osteoclasts exhibit unique phenotypic traits, including increased apoptosis and defective early stage autophagy. Intracellular and extracellular acidification is absent in R740S/R740S osteoclasts, providing evidence for a requirement for lysosomal acidification for cytoplasmic distribution of key osteoclast enzymes such as TRAP and other important osteoclast phenotypic traits. This work provides evidence that the a3 subunit of V-ATPases and the proton pumping function of a3-containing V-ATPases play a major role in osteoclast survival, maturation and function.