An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells

Abstract

SUMMARYVacuolar sorting receptors (VSRs) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the pre-vacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here we developed and tested an in vivo expression system for the identification of VSR cargos which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture medium along with their corresponding cargo proteins. Indeed, transgenic Arabidopsis culture cell lines expressing VSR N-terminal binding domains (VSRNTs) were shown to secrete truncated VSRs (BP80NT, AtVSR1NT and AtVSR4NT) with attached cargo molecules into the culture medium. Putative cargo proteins were identified through mass spectrometry. Several identified cargo proteins were confirmed by localization studies and interaction analysis with VSRs. The screening strategy described here should be applicable to all VSRs and will help identify and study cargo proteins for individual VSR proteins. This method should be useful for both cargo identification and protein-protein interaction in vivo.

Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis

“…Confocal images were collected at 12 to 14 h or 36 to 48 h after transformation as indicated using an Olympus FV1000 system. Images were processed using Adobe Photoshop as described previously (Jiang and Rogers, 1998;Shen et al, 2013a). Time-lapse movie and images were obtained by spinning disc confocal microscopy (Andor) and analyzed by Imaris software (Bitplane) as described previously (Wang et al, 2010;Ding et al, 2014).…”

Trans-Golgi Network-Located AP1 Gamma Adaptins Mediate Dileucine Motif-Directed Vacuolar Targeting in Arabidopsis

“…Notably, GFP-VIT1 also showed punctate fluorescent signals in addition to the tonoplast. To verify the identity of these punctae, we coexpressed GFP-VIT1 with known organelle markers in Arabidopsis protoplasts, such as the Golgi marker ManI-RFP, the TGN marker RFP-SYP61, and the PVC marker RFP-VSR2 (Shen et al, 2013;Cai et al, 2014). The punctate signal of GFP-VIT1 was clearly separate from ManI-RFP ( Figure 1E), while it showed partially colocalization with the TGN marker RFP-SYP61 (Figure 1F) and with the PVC marker RFP-VSR2 ( Figure 1G).…”

How Vacuolar Sorting Receptor Proteins Interact with Their Cargo Proteins: Crystal Structures of Apo and Cargo-Bound Forms of the Protease-Associated Domain from an Arabidopsis Vacuolar Sorting Receptor

“…In the complex structure, the C-terminal tail is anchored to the PA domain by a hydrogen bond between two highly conserved residues, Glu-24 and His-181 ( Figure 5A). We anticipated that the double mutations, E24A/H181A, will break the hydrogen bond, destabilize the bound (A) Construct of VSR1NT-T7, NT region of Arabidopsis VSR1 with its native signal peptide (SP), and a C-terminal T7 tag (Shen et al, 2013).…”

“…(B) Constructs used for aleu-VSD-GFP and its variants: aleu-VSD-GFP, previously described VSD from barley aleurain (Humair et al, 2001;Holwerda et al, 1992) following its native signal peptide; S3G-aleu-VSD-GFP and I6G-aleu-VSD-GFP, mutants of aleu-VSD-GFP, introduced mutation are underlined; Sec-GFP, the secreted form of GFP in which the signal peptide of sweet potato (Ipomoea batatas) sporamin was fused to GFP (Shen et al, 2013).…”

“…BP-80 from pea (Pisum sativum) was the first VSR protein identified, which binds to a NPIR-containing sequence motif located at the N-terminal region of aleurain (Kirsch et al, 1994;Paris and Neuhaus, 2002;Paris et al, 1997;Humair et al, 2001). The NPIR-containing sequence motif is the most well studied VSD and is found in a number of vacuolar targeting proteins Nakamura, 1991, 1999;Holwerda et al, 1992;Kirsch et al, 1994Kirsch et al, , 1996Paris et al, 1997;Ahmed et al, 2000;Humair et al, 2001;Watanabe et al, 2002Watanabe et al, , 2004Shimada et al, 2003;Suen et al, 2010;Shen et al, 2013). Mutagenesis studies have revealed that receptor-cargo recognition is sequence specific (Matsuoka et al, 1995;Kirsch et al, 1996;Shimada et al, 1997;Matsuoka and Nakamura, 1999;Suen et al, 2010).…”