Cláudia Pereira

Exploring plant endomembrane dynamics using the photoconvertible protein Kaede

Photoactivatable and photoconvertible fluorescent proteins capable of pronounced light-induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse-chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl-transferase (ST-Kaede) and of the vacuolar pathway marker cardosin A (cardA-Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede-based imaging protocols. Photoconverted ST-Kaede red-labelled organelles can be followed within neighbouring populations of non-converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio-imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack.

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases

Summary.Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied. mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development.

Molecular mechanism of dynein recruitment to kinetochores by the Rod–Zw10–Zwilch complex and Spindly

The molecular motor dynein concentrates at the kinetochore region of mitotic chromosomes in animals to accelerate spindle microtubule capture and to control spindle checkpoint signaling. In this study, we describe the molecular mechanism used by the Rod–Zw10–Zwilch complex and the adaptor Spindly to recruit dynein to kinetochores in Caenorhabditis elegans embryos and human cells. We show that Rod’s N-terminal &bgr;-propeller and the associated Zwilch subunit bind Spindly’s C-terminal domain, and we identify a specific Zwilch mutant that abrogates Spindly and dynein recruitment in vivo and Spindly binding to a Rod &bgr;-propeller–Zwilch complex in vitro. Spindly’s N-terminal coiled-coil uses distinct motifs to bind dynein light intermediate chain and the pointed-end complex of dynactin. Mutations in these motifs inhibit assembly of a dynein–dynactin–Spindly complex, and a null mutant of the dynactin pointed-end subunit p27 prevents kinetochore recruitment of dynein–dynactin without affecting other mitotic functions of the motor. Conservation of Spindly-like motifs in adaptors involved in intracellular transport suggests a common mechanism for linking dynein to cargo.

Structure-activity relationships for dipeptide prodrugs of acyclovir: implications for prodrug design

A series of water-soluble dipeptide ester prodrugs of the antiviral acyclovir (ACV) were evaluated for their chemical stability, cytotoxicity, and antiviral activity against several strains of Herpes Simplex-1 and -2, vaccinia, vesicular stomatitis, cytomegalovirus and varicella zoster viruses. ACV dipeptide esters were very active against herpetic viruses, independently of the rate at which they liberate the parent drug. Their minimum cytotoxic concentrations were above 100 microM and the resulting MCC/EC(50) values were lower than those of ACV. When comparing the reactivity of Phe-Gly esters and amides (ACV, zidovudine, paracetamol, captopril and primaquine) in pH 7.4 buffer it was found that the rate of drug release increases with drugs leaving group ability. Release of the parent drug from Phe-Gly in human plasma is markedly faster than in pH 7.4 buffer, thus suggesting that the dipeptide-based prodrug approach can be successfully applied to bioactive agents containing thiol, phenol and amine functional groups.

Delivering of Proteins to the Plant Vacuole—An Update

Trafficking of soluble cargo to the vacuole is far from being a closed issue as it can occur by different routes and involve different intermediates. The textbook view of proteins being sorted at the post-Golgi level to the lytic vacuole via the pre-vacuole or to the protein storage vacuole mediated by dense vesicles is now challenged as novel routes are being disclosed and vacuoles with intermediate characteristics described. The identification of Vacuolar Sorting Determinants is a key signature to understand protein trafficking to the vacuole. Despite the long established vacuolar signals, some others have been described in the last few years, with different properties that can be specific for some cells or some types of vacuoles. There are also reports of proteins having two different vacuolar signals and their significance is questionable: a way to increase the efficiency of the sorting or different sorting depending on the protein roles in a specific context? Along came the idea of differential vacuolar sorting, suggesting a possible specialization of the trafficking pathways according to the type of cell and specific needs. In this review, we show the recent advances in the field and focus on different aspects of protein trafficking to the vacuoles.

Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors.

The kinetochore-specific dynein adaptor Spindly is identified as a novel substrate of farnesyltransferase in human cells. Farnesylation is required for Spindly accumulation at kinetochores, and nonfarnesylated Spindly delays chromosome congression, providing new mechanistic insight into the biological effect of farnesyltransferase inhibitors.

Cardosin A contains two vacuolar sorting signals using different vacuolar routes in tobacco epidermal cells

Several vacuolar sorting determinants (VSDs) have been described for protein trafficking to the vacuoles in plant cells. Because of the variety in plant models, cell types and experimental approaches used to decipher vacuolar targeting processes, it is not clear whether the three well-known groups of VSDs identified so far exhaust all the targeting mechanisms, nor if they reflect certain protein types or families. The vacuolar targeting mechanisms of the aspartic proteinases family, for instance, are not yet fully understood. In previous studies, cardosin A has proven to be a good reporter for studying the vacuolar sorting of aspartic proteinases. We therefore propose to explore the roles of two different cardosin A domains, common to several aspartic proteinases [i.e. the plant-specific insert (PSI) and the C-terminal peptide VGFAEAA] in vacuolar sorting. Several truncated versions of the protein conjugated with fluorescent protein were made, with and without these putative sorting determinants. These domains were also tested independently, for their ability to sort other proteins, rather than cardosin A, to the vacuole. Fluorescent chimaeras were tracked in vivo, by confocal laser scanning microscopy, in Nicotiana tabacum cells. Results demonstrate that either the PSI or the C terminal was necessary and sufficient to direct fluorescent proteins to the vacuole, confirming that they are indeed vacuolar sorting determinants. Further analysis using blockage experiments of the secretory pathway revealed that these two VSDs mediate two different trafficking pathways.

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

Organ-specific distribution and subcellular localisation of ascorbate peroxidase isoenzymes in potato (Solanum tuberosum L.) plants

Summary.Ascorbate peroxidase (EC 1.11.1.11), a heme-containing homodimeric protein, is a hydrogen peroxide-scavenging enzyme, playing an important role in plants in order to protect them from oxidative stress, thus adverting cellular damage. Several ascorbate peroxidase isoenzymes have been reported but the understanding of their physiological role still depends on a better knowledge of their precise localisation within plant organs. Immunocytochemistry techniques were performed in order to elucidate the peroxisomal and cytosolic ascorbate peroxidase distribution within tissues of leaves and sprouts of potato plants. The peroxisomal isoenzyme was found to have a broad distribution in sprouts, but a differential one in leaves, being restricted to the spongy parenchyma. This differential expression may be associated to the mesophyll asymmetry and the diverse physiological processes that occur in it. The cytosolic isoenzyme was not detected in leaves under the used conditions, probably because it is present in low amounts in these tissues. The results obtained in sprouts were at least curious: cytosolic ascorbate was found to be adjacent to the amyloplasts. Given these results, it is possible to state that apart from their similarity, these two isoenzymes reside in different organelles and seem to take part in different physiological processes as suggested by their organ- and tissue-specific distribution.

Isolation and characterisation of a cDNA encoding a novel cytosolic ascorbate peroxidase from potato plants (Solanum tuberosum L.)

Ascorbate peroxidase (APX) is one of the key enzymes of the plant antioxidant system playing, along with catalase, a central role in hydrogen peroxide scavenging. An approach to further increase the knowledge about cytosolic APX gene organization can be achieved by isolating and characterisating new cDNAs, thus providing new insights about the physiological roles and regulation of these enzymes. A partial cDNA clone (corresponding to the 3’ untranslated region), cytosolic ascorbate peroxidase-related, was isolated from potato sprouts by RT-PCR. Database analysis retrieved several expressed sequence tags (ESTs) coding potato cytosolic ascorbate peroxidase, that were used to infer the complete cDNA sequence. The deduced amino acid sequence revealed high homologies with other plant cytosolic ascorbate peroxidases, confirming the reliability of the virtual cDNA. Northern blot analysis revealed the existence of a single band related to the isolated cDNA and the southern blotting results allowed the elaboration of a possible gene organization.