Abstract
In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis βFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.
Highlights
In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments
In vsr1, vsr3, and vsr4 mutant protoplasts, the degree of AALP: GFP secretion varies depending on these vsr mutants, a small proportion (3%–9%) of AALP:GFP was secreted into the incubation medium and the amount of the
AtCPY was secreted into the incubation medium when transiently expressed in vsr1vsr4 mutant protoplasts, but not in wild-type protoplasts (Supplemental Figure S2). These results indicate that VSR1, VSR3, and VSR4, but not VSR5 and VSR6, are involved in vacuolar trafficking of sequence-specific vacuolar sorting signal (ssVSS)-containing proteins in vegetative tissues, and they are functionally redundant in protein trafficking to the lytic vacuole (LV)
Summary
Protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. Of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr protoplasts Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells. One of them is the lytic vacuole (LV) that is present in vegetative cells, and the other is the protein storage vacuole (PSV) that is present in seed cells (Frigerio et al, 2008; Zouhar and Rojo, 2009; De Marcos Lousa et al, 2012) These two types of vacuoles have different functions. The luminal domain but not the cytosolic tail of VSRs contains the determinant for the sorting specificity
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