The tyrosine-sorting motif of the vacuolar sorting receptor VSR4 from Arabidopsis thaliana, which is involved in the interaction between VSR4 and AP1M2, μ1-adaptin type 2 of clathrin adaptor complex 1 subunits, participates in the post-Golgi sorting of VSR4

Abbreviations: Amp^r, ampicillin resistance marker; AP, adaptor protein; AP1M, μ1-adaptin of AP1 complex; AP1M2, AP1M type 2; BiFC, bimolecular fluorescence complementation; BP, a recombination between an att B-flanking DNA fragment and an att P-containing donor vector; CaMV, cauliflower mosaic virus; CDS, coding sequence; CCV, clathrin-coated vesicle; CLSM, confocal laser scanning microscope; COPI, coat protein I; COPII, coat protein II; CRS, cargo-recognition sequence; cV, C-terminal region (211–239) of Venus; ECFP, enhanced cyan fluorescent protein; EE, early endosome; ER, endoplasmic reticulum; GFP, green fluorescent protein; IP, immunoprecipitation; Km^r, kanamycin resistance marker; mCherry, monomeric Cherry; nV, N-terminal region (1–210) of Venus; ORF, open reading frame; P_35S, 35S promoter; LR, a recombination between an att L-containing entry clone and an att R-containing destination vector; PCR, polymerase chain reaction; PM, plasma membrane; PPI, protein–protein interaction; PVDF, polyvinylidene fluoride; RFP, red fluorescent protein; RME, receptor-mediated endocytosis; SD, standard deviation; SDS-PAGE, SDS‐polyacrylamide gel electrophoresis; sGFP, synthetic GFP with S65T; SYP, syntaxin of plants; tdTomato, tandem dimer Tomato; TGN, trans-Golgi network; VM, vacuolar membrane; VSR, vacuolar-sorting receptor; YFP, yellow fluorescent protein.

Abstract

μ1-Adaptin of adaptor protein (AP) 1 complex, AP1M, is generally accepted to load cargo proteins into clathrin-coated vesicles (CCVs) at the trans-Golgi network through its binding to cargo-recognition sequences (CRSs). Plant vacuolar-sorting receptors (VSRs) function in sorting vacuolar proteins, which are reportedly mediated by CCV. We herein investigated the involvement of CRSs of Arabidopsis thaliana VSR4 in the sorting of VSR4. The results obtained showed the increased localization of VSR4 at the plasma membrane or vacuoles by mutations in CRSs including the tyrosine-sorting motif YMPL or acidic dileucine-like motif EIRAIM, respectively. Interaction analysis using the bimolecular fluorescence complementation (BiFC) system, V10-BiFC, which we developed, indicated an interaction between VSR4 and AP1M2, AP1M type 2, which was attenuated by a YMPL mutation, but not influenced by an EIRAIM mutation. These results demonstrated the significance of the recognition of YMPL in VSR4 by AP1M2 for the post-Golgi sorting of VSR4.

Graphical abstract

Clathrin AP1M2-recognizable tyrosine motif for plant VSR4 sorting.

Key words:

• Arabidopsis thaliana
• BiFC
• clathrin
• VSR4
• tyrosine-sorting motif

Acknowledgments

We thank Dr. Takashi Ueda at the University of Tokyo, Dr. Roger Tsien at the University of California, and Dr. Shoji Mano at the National Institute for Basic Biology for the gift of cDNAs of organellar marker genes, red fluorescence proteins, such as tdTomato and mCherry, and destination vectors, including pB5tdGW and pB5mCGW, respectively. We are also grateful to Dr. Hideyuki Yamaguchi of Leica Microsystems for the quantification of fluorescence images.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes

Abbreviations: Amp^r, ampicillin resistance marker; AP, adaptor protein; AP1M, μ1-adaptin of AP1 complex; AP1M2, AP1M type 2; BiFC, bimolecular fluorescence complementation; BP, a recombination between an att B-flanking DNA fragment and an att P-containing donor vector; CaMV, cauliflower mosaic virus; CDS, coding sequence; CCV, clathrin-coated vesicle; CLSM, confocal laser scanning microscope; COPI, coat protein I; COPII, coat protein II; CRS, cargo-recognition sequence; cV, C-terminal region (211–239) of Venus; ECFP, enhanced cyan fluorescent protein; EE, early endosome; ER, endoplasmic reticulum; GFP, green fluorescent protein; IP, immunoprecipitation; Km^r, kanamycin resistance marker; mCherry, monomeric Cherry; nV, N-terminal region (1–210) of Venus; ORF, open reading frame; P_35S, 35S promoter; LR, a recombination between an att L-containing entry clone and an att R-containing destination vector; PCR, polymerase chain reaction; PM, plasma membrane; PPI, protein–protein interaction; PVDF, polyvinylidene fluoride; RFP, red fluorescent protein; RME, receptor-mediated endocytosis; SD, standard deviation; SDS-PAGE, SDS‐polyacrylamide gel electrophoresis; sGFP, synthetic GFP with S65T; SYP, syntaxin of plants; tdTomato, tandem dimer Tomato; TGN, trans-Golgi network; VM, vacuolar membrane; VSR, vacuolar-sorting receptor; YFP, yellow fluorescent protein.