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. 2007 Dec;81(23):12881-8.
doi: 10.1128/JVI.00913-07. Epub 2007 Sep 26.

HC-Pro protein of Potato virus Y can interact with three Arabidopsis 20S proteasome subunits in planta

Yongsheng Jin  1 , Dongyuan Ma, Jiangli Dong, Jingchen Jin, Daofeng Li, Changwang Deng, Tao Wang
Affiliations

HC-Pro protein of Potato virus Y can interact with three Arabidopsis 20S proteasome subunits in planta

Yongsheng Jin et al. J Virol. 2007 Dec.

Abstract

The multifunctional protein helper component proteinase (HC-Pro) is thought to interfere with the activity of the 20S proteasome; however, no sites of interaction have been identified for either protein. Here, we first show that the Potato virus Y (PVY) HC-Pro protein can interact with three Arabidopsis 20S proteasome subunits (PAA, PBB, and PBE), using a yeast two-hybrid system and the bimolecular fluorescence complement assay. In addition, yeast two-hybrid analysis of the interaction between several mutant subunits of the 20S proteasome and PVY HC-Pro confirmed that residues 81 to 140 of PAA, 1 to 80 of PBB, and 160 to 274 of PBE are necessary for binding PAA, PBB, and PBE to PVY HC-Pro, respectively. Deletion mutant analysis of PVY HC-Pro showed that the N terminus (residues 1 to 97) is necessary for its interaction with three Arabidopsis 20S proteasome subunits. The ability of HC-Pro to interact and interfere with the activity of the 20S proteasome may help explain the molecular basis of its multifunctional character.

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Figures

FIG. 1.
FIG. 1.
Interaction of 14 Arabidopsis 20S proteasome subunits and PVY HC-Pro in transformed S. cerevisiae AH109 cells grown on SD/−Leu/−Trp (a) and on SD/−Ade/−His/−Leu/−Trp (b). Cotransformants of seven a subunits (left) and seven β subunits (right) (PAA to PAE) and HC-Pro are shown. +, pGBKT7-53/pGADT7-RecT (positive control); −, pGBKT7-HC-Pro/pGADT7 (negative control); 1, pGBKT7-HC-Pro/pGADT7-PAA; 2, pGBKT7-HC-Pro/pGADT7-PAB; 3, pGBKT7-HC-Pro/pGADT7-PAC; 4, pGBKT7-HC-Pro/pGADT7-PAD; 5, pGBKT7-HC-Pro/pGADT7-PAE; 6, pGBKT7-HC-Pro/pGADT7-PAF; 7, pGBKT7-HC-Pro/pGADT7-PAG; 8, pGBKT7-HC-Pro/pGADT7-PBA; 9, pGBKT7-HC-Pro/pGADT7-PBB; 10, pGBKT7-HC-Pro/pGADT7-PBC; 11, pGBKT7-HC-Pro/pGADT7-PBD; 12, pGBKT7-HC-Pro/pGADT7-PBE; 13, pGBKT7-HC-Pro/pGADT7-PBF; 14, pGBKT7-HC-Pro/pGADT7-PBG.
FIG. 2.
FIG. 2.
Fluorescence of reconstructed YFP complexes (green) in onion (A. cepa) epidermal cells.
FIG. 3.
FIG. 3.
Subcellular localization of reconstructed YFP complexes determined in leaf epidermis of N. benthamiana. a, YFP fluorescence (green); b, chlorophyll autofluorescence (red); c, bright field; d, YFP-chlorophyll autofluorescence overlay.
FIG. 4.
FIG. 4.
Schematic overview of domains and deletion mutants of PAA (A), PBB (B), and PBE (C).
FIG. 5.
FIG. 5.
Interaction of PVY HC-Pro and mutants of PAA (left), PBB (middle), and PBE (right) in transformed S. cerevisiae AH109 cells grown on SD/−Leu/−Trp (a) and on SD/−Ade/−His/−Leu/−Trp (b). +, pGBKT7-53/pGADT7-RecT (positive control); −, pGBKT7-HC-Pro/pGADT7 (negative control); 1, pGBKT7-HC-Pro/pGADT7-PAA; 2, pGBKT7-HC-Pro/pGADT7-PAA1; 3, pGBKT7-HC-Pro/pGADT7-PAA2; 4, pGBKT7-HC-Pro/pGADT7-PAA3; 5, pGBKT7-HC-Pro/pGADT7-PBB; 6, pGBKT7-HC-Pro/pGADT7-PBB1; 7, pGBKT7-HC-Pro/pGADT7-PBB2; 8, pGBKT7-HC-Pro/pGADT7-PBE; 9, pGBKT7-HC-Pro/pGADT7-PBE1; 10, pGBKT7-HC-Pro/pGADT7-PBE2.
FIG. 6.
FIG. 6.
Schematic overview of domains and deletion mutants of PVY HC-Pro.
FIG. 7.
FIG. 7.
Interaction of mutants of PVY HC-Pro and PAA, PBB, and PBE in transformed S. cerevisiae AH109 cells grown on SD/−Leu/−Trp (a) and on SD/−Ade/−His/−Leu/−Trp (b). Cotransformants of HC-Pro1 (left), HC-Pro2 (middle), and HC-Pro3 (right) and PAA, PBB, and PBE are shown. +, pGBKT7-53/pGADT7-RecT (positive control); −, pGBKT7-HC-Pro/pGADT7 (negative control); 1, pGBKT7-HC-Pro1/pGADT7-PAA; 2, pGBKT7-HC-Pro1/pGADT7-PBB; 3, pGBKT7-HC-Pro1/pGADT7-PBE; 4, pGBKT7-HC-Pro2/pGADT7-PAA; 5, pGBKT7-HC-Pro2/pGADT7-PBB; 6, pGBKT7-HC-Pro2/pGADT7-PBE; 7, pGBKT7-HC-Pro3/pGADT7-PAA; 8, pGBKT7-HC-Pro3/pGADT7-PBB; 9, pGBKT7-HC-Pro3/pGADT7-PBE.
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