The secretory pathway of plants is a highly complex system concerned with the manufacture, quality control and transport of various polymers. Errors in transcription, translation and inefficient folding are among the many factors that can contribute to the accumulation of misfolded or unassembled polypeptides within the secretory pathway. These proteins are potentially very damaging and must therefore be eliminated. This was initially assumed to involve degradation in lytic vacuoles following targeting or autophagy. However, it was later demonstrated that the plant endoplasmic reticulum (ER) is endowed with a system that allows the retrotranslocation of terminally misfolded or orphan proteins to the cytosol where these selected substrates can then be degraded1. This quality control pathway known as ER-associated protein degradation (ERAD) is well described in mammalian cells, where it typically involves delivery of the selected proteins to dislocation machineries in the membrane, substrate recognition on the cytosolic plane of the membrane brought about by the ubiquitination machinery, extraction from the membrane and degradation by proteasomes. These events are tightly coupled such that it is normally very difficult to visualise a retrotranslocated substrate unless proteasomes are inhibited. Although several different ERAD substrates have now been described in plants, so far we are almost completely ignorant not only of the range of plant proteins that undergo ERAD, but of how many endogenous proteins are devoted to the disposal of ER proteins. Of note, over 1300 genes (5% of the Arabidopsis proteome) are devoted to the selective breakdown of proteins by the extensive ubiquitin/26S proteasome pathway in plant cells, but virtually nothing is known about these components in relation to ERAD, nor of the overall impact of this pathway relative to alternative disposal mechanisms. Addressing these important questions until now has been extremely difficult due to the limited information concerning both the biochemical properties of plant proteasomes and the activity of proteasome inhibitors in living plant cells. For this reason we characterized the cleavage specificities of proteasomes in protoplasts extracts of Nicotiana tabacum using fluorogenic substrates specific for the main peptidase activities of 26S particles. Furthermore, we evaluated the in vitro sensibility of these activities to several reversible and irreversible inhibitors widely used to inhibit mammalian and yeast proteasomes. Finally we measured the extent of proteasomal inhibition reached in vivo by administration of these inhibitors to protoplasts cultures. Based upon these results, we plan to develop more effective protocols of proteasomal in vitro and in vivo inhibition that will facilitate further study on ERAD in plants cells.
The role of ERAD in plant cells: a preliminary biochemical characterization of plant proteasomes
CASCIO, Paolo;CERRUTI, Fulvia;
2008-01-01
Abstract
The secretory pathway of plants is a highly complex system concerned with the manufacture, quality control and transport of various polymers. Errors in transcription, translation and inefficient folding are among the many factors that can contribute to the accumulation of misfolded or unassembled polypeptides within the secretory pathway. These proteins are potentially very damaging and must therefore be eliminated. This was initially assumed to involve degradation in lytic vacuoles following targeting or autophagy. However, it was later demonstrated that the plant endoplasmic reticulum (ER) is endowed with a system that allows the retrotranslocation of terminally misfolded or orphan proteins to the cytosol where these selected substrates can then be degraded1. This quality control pathway known as ER-associated protein degradation (ERAD) is well described in mammalian cells, where it typically involves delivery of the selected proteins to dislocation machineries in the membrane, substrate recognition on the cytosolic plane of the membrane brought about by the ubiquitination machinery, extraction from the membrane and degradation by proteasomes. These events are tightly coupled such that it is normally very difficult to visualise a retrotranslocated substrate unless proteasomes are inhibited. Although several different ERAD substrates have now been described in plants, so far we are almost completely ignorant not only of the range of plant proteins that undergo ERAD, but of how many endogenous proteins are devoted to the disposal of ER proteins. Of note, over 1300 genes (5% of the Arabidopsis proteome) are devoted to the selective breakdown of proteins by the extensive ubiquitin/26S proteasome pathway in plant cells, but virtually nothing is known about these components in relation to ERAD, nor of the overall impact of this pathway relative to alternative disposal mechanisms. Addressing these important questions until now has been extremely difficult due to the limited information concerning both the biochemical properties of plant proteasomes and the activity of proteasome inhibitors in living plant cells. For this reason we characterized the cleavage specificities of proteasomes in protoplasts extracts of Nicotiana tabacum using fluorogenic substrates specific for the main peptidase activities of 26S particles. Furthermore, we evaluated the in vitro sensibility of these activities to several reversible and irreversible inhibitors widely used to inhibit mammalian and yeast proteasomes. Finally we measured the extent of proteasomal inhibition reached in vivo by administration of these inhibitors to protoplasts cultures. Based upon these results, we plan to develop more effective protocols of proteasomal in vitro and in vivo inhibition that will facilitate further study on ERAD in plants cells.
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