Abstract
Ribosome-inactivating proteins (RIPs) are cytotoxic enzymes that inhibit protein translation by depurinating ribosomal RNA. Although most plant RIPs are synthesized with leader sequences that sequester them away from the host ribosomes, several RIPs from cereals lack these signal peptides and therefore probably reside in the cytosol near the plant ribosomes. More than 30 RIP genes have been identified in the rice (Oryza sativa spp. japonica) genome, many of them lacking a signal peptide. This paper focuses on a presumed cytosolic type-1 RIP from rice, referred to as OsRIP1. Using 3D modeling it is shown that OsRIP1 structurally resembles other cereal RIPs and has an active site that meets the requirements for activity. Furthermore, localization studies indicate that OsRIP1-eGFP fusion proteins reside in the nucleocytoplasmic space when expressed in epidermal cells of Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Finally, OsRIP1 was recombinantly produced in Escherichia coli and was demonstrated to possess catalytic activity. Interestingly, this recombinant RIP inactivates wheat ribosomes far less efficiently than rabbit ribosomes in an in vitro system. These findings raise some interesting questions concerning the mode of action and physiological role of OsRIP1. This is the first time a RIP from rice is investigated at protein level and is shown to possess biological activity.
Highlights
Ribosome-inactivating proteins (RIPs) are enzymes that can irreversibly inhibit protein translation by removing a specific adenine residue from the conserved sarcin/ricin loop of the 28S rRNA
This alignment revealed that the active site residues (Y79, Y112, E167, R170, and W204) that make up the specificity pocket of RIPs are conserved in OsRIP1
OsRIP1 shows the highest degree of sequence identity to the barley barley RIP1 (bRIP1) (57.9%), followed by the barley bRIP2 (57.1%) and tritin (56.8%)
Summary
Ribosome-inactivating proteins (RIPs) are enzymes that can irreversibly inhibit protein translation by removing a specific adenine residue from the conserved sarcin/ricin loop of the 28S rRNA. The toxic effect of RIPs towards host cells is hypothesized to represent a defense mechanism by which damaged plant cells are eliminated through apoptosis after release of the sequestered RIPs in the cytosol [15,16,17] Next to these classical RIPs, some RIP lineages appear to be synthesized without signal peptides and are suggested to reside in the cytosolic compartment where they can come into contact with the host ribosomes [18,19,20,21]. In the rice genome both RIP genes containing a signal peptide as well as RIP genes lacking signal sequences have been identified [28], suggesting that rice plants express both cytosolic and secreted type-1 RIPs. This study investigates the structure, subcellular localization and catalytic activity of OsRIP1, a presumed cytosolic type-1 RIP from Oryza sativa. We demonstrate that OsRIP1, recombinantly produced in E. coli, inhibits translation and is far less active on cereal ribosomes compared to animal ribosomes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
