RESUMO
Numerous proteins are transported into or across the chloroplast thylakoid membrane. To date, two major pathways have been identified for the transport of luminal proteins (the Sec- and Tat-dependent pathways) and it is now clear that these protein translocases use fundamentally different transport mechanisms. Integral membrane proteins are inserted by means of at least two further pathways. One involves the input of numerous targeting factors, including SRP (signal recognition particle), FtsY and Albino3. Surprisingly, the other pathway does not involve any of the known chloroplastic targeting factors, and insertion is energy-independent, raising the possibility of an unusual 'spontaneous' insertion mechanism.
Assuntos
Tilacoides/metabolismo , Membranas Intracelulares/metabolismo , Modelos Biológicos , Proteínas de Plantas/metabolismo , Transporte ProteicoRESUMO
When expressed in tobacco cells, the catalytic subunit of the dimeric ribosome inactivating protein, ricin, is first inserted into the endoplasmic reticulum (ER) and then degraded in a manner that can be partially inhibited by the proteasome inhibitor clasto-lactacystin beta-lactone. Consistent with the implication of cytosolic proteasomes, degradation of ricin A chain is brefeldin A-insensitive and the polypeptides that accumulate in the presence of the proteasome inhibitor are not processed in a vacuole-specific fashion. Rather, these stabilized polypeptides are in part deglycosylated by a peptide:N-glycanase-like activity. Taken together, these results indicate that ricin A chain, albeit a structurally native protein, can behave as a substrate for ER to cytosol export, deglycosylation in the cytosol, and proteasomal degradation. Furthermore, retrotranslocation of this protein is not tightly coupled to proteasomal activity. These data are consistent with the hypothesis that ricin A chain can exploit the ER-associated protein degradation pathway to reach the cytosol. Although well characterized in mammalian and yeast cells, the operation of a similar pathway to the cytosol of plant cells has not previously been demonstrated.
Assuntos
Nicotiana/metabolismo , Ricina/metabolismo , Transporte Biológico Ativo , Brefeldina A/farmacologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Lactonas/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Lectinas de Plantas , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma , Subunidades Proteicas , Ricina/química , Ricina/genética , Nicotiana/citologia , Nicotiana/genéticaRESUMO
Ricin is a heterodimeric toxin that accumulates in the storage vacuoles of castor bean (Ricinus communis) endosperm. Proricin is synthesized as a single polypeptide precursor comprising the catalytic A chain and the Gal-binding B chain joined by a 12-amino acid linker propeptide. Upon arrival in the vacuole, the linker is removed. Here, we replicate these events in transfected tobacco (Nicotiana tabacum) leaf protoplasts. We show that the internal linker propeptide is responsible for vacuolar sorting and is sufficient to redirect the ricin heterodimer to the vacuole when fused to the A or the B chain. This internal peptide can also target two different secretory protein reporters to the vacuole. Moreover, mutation of the isoleucine residue within an NPIR-like motif of the propeptide affects vacuolar sorting in proricin and in the reconstituted A-B heterodimer. This is the first reported example of a sequence-specific vacuolar sorting signal located within an internal propeptide.
Assuntos
Endocitose , Ricina/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Lectinas de Plantas , Plantas Tóxicas , Ricina/química , Homologia de Sequência de Aminoácidos , Nicotiana/metabolismo , Nicotiana/ultraestruturaRESUMO
We report here the successful establishment of callus, cell and root cultures from explants of in-vitro-grown plantlets of the soapwort Saponaria officinalis L. The production of saporin in the different tissue systems was evaluated by determining the capability of crude extracts to inactivate protein synthesis and by Western blotting analysis. Protein synthesis inhibition varied in callus and derived cell suspensions and in cultured roots, the latter, in particular, showing the lowest specific activity. The ribosome-inactivating principle from root cultures was purified to homogeneity by cation exchange chromatography.
RESUMO
The pattern of expression of the rolB gene, derived from the T-DNA of the plant pathogen Agrobacterium rhizogenes, has been investigated during the early stages of somatic embryo formation in suspension cultures of carrot (Daucus carota L.). The reporter gene GUS (Escherichia coli beta-glucuronidase), under transcriptional control of full-length rolB promoter region, has been utilized in order to evaluate both qualitative and quantitative variations in the expression pattern. Fluorimetric measurements point to the developmental regulation of the gene, while results from histochemical analysis indicate that the promoter of rolB is firstly activated in the central (core) region of the globular embryos.