Abstract
In Arabidopsis thaliana, different types of vacuolar receptors were discovered. The AtVSR (Vacuolar Sorting Receptor) receptors are well known to be involved in the traffic to lytic vacuole (LV), while few evidences demonstrate the involvement of the receptors from AtRMR family (Receptor Membrane RING-H2) in the traffic to the protein storage vacuole (PSV). In this study we focused on the localization of two members of AtRMR family, AtRMR1 and -2, and on the possible interaction between these two receptors in the plant secretory pathway. Our experiments with agroinfiltrated Nicotiana benthamiana leaves demonstrated that AtRMR1 was localized in the endoplasmic reticulum (ER), while AtRMR2 was targeted to the trans-Golgi network (TGN) due to the presence of a cytosolic 23-amino acid sequence linker. The fusion of this linker to an equivalent position in AtRMR1 targeted this receptor to the TGN, instead of the ER. By using a Bimolecular Fluorescent Complementation (BiFC) technique and experiments of co-localization, we demonstrated that AtRMR2 can make homodimers, and can also interact with AtRMR1 forming heterodimers that locate to the TGN. Such interaction studies strongly suggest that the transmembrane domain and the few amino acids surrounding it, including the sequence linker, are essential for dimerization. These results suggest a new model of AtRMR trafficking and dimerization in the plant secretory pathway.
Highlights
Vacuolar proteins are targeted to the different types of vacuoles via organelles that communicate with each other by a highly regulated process involving different kinds of vesicles as well as cisternal maturation [1,2]
We demonstrated by Bimolecular Fluorescent Complementation (BiFC) that AtRMR2 forms homodimers and can interact with AtRMR1 to form heterodimers, which localizes to the trans-Golgi network (TGN)
The aforementioned experiments of BiFC and co-localization using deletion mutants strongly suggest that the main protein domains of AtRMRs (PA, RING-H2 and Ser-Rich domains) are not essential for protein-protein interaction and that the transmembrane domain (TM) and/or the flanking luminal and cytosolic linkers are involved in protein dimerization
Summary
Vacuolar proteins are targeted to the different types of vacuoles via organelles that communicate with each other by a highly regulated process involving different kinds of vesicles as well as cisternal maturation [1,2]. We demonstrated by Bimolecular Fluorescent Complementation (BiFC) that AtRMR2 forms homodimers and can interact with AtRMR1 to form heterodimers, which localizes to the TGN These experiments performed using AtRMR deletion mutants of different cytosolic (PA) and luminal (RING-H2 and Ser-Rich) domains, suggest that only the transmembrane domain and the neighboring sequences, including the sequence linker, are necessary for this dimerization. .InHtohwisecvaeser,, aa cploesairtivsiegnal was saplsliot-YobFPsesrivgendalwwhaesnobcsoe-revxepdreinsspiunngcAtattRe MstrRu2ct∆uRreisn,gwSheirleanERd lAoctRalMizaRti1o∆nRoifnAgtfRuMseRd1∆toRicnogm-RpFlPemweanstary YFP fragments (Figure 8e,f) supporting the ability of these two AtRMR receptors to produce heterodimers Such experiments support previous results of AtRMRs co-localization and the evidence that the TM and/or the amino acids surrounding it, including L, are the domains involved in AtRMR dimerization. The expression in N. benthamiana leaves of AtRMR1 and AtRMR2 deletion mutants lacking the PA, RING-H2 or Ser-Rich domains clearly showed that all these mutants had the same localization as the full-length wild-type proteins (Figure 4). Other signals and/or mechanisms must determine the subcellular localization of these receptors
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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
