Significant role of PB1 and UBA domains in multimerization of Joka2, a selective autophagy cargo receptor from tobacco

Abstract

Tobacco Joka2 protein is a hybrid homolog of two mammalian selective autophagy cargo receptors, p62 and NBR1. These proteins can directly interact with the members of ATG8 family and the polyubiquitinated cargoes designed for degradation. Function of the selective autophagy cargo receptors relies on their ability to form protein aggregates. It has been shown that the N-terminal PB1 domain of p62 is involved in formation of aggregates, while the UBA domains of p62 and NBR1 have been associated mainly with cargo binding. Here we focus on roles of PB1 and UBA domains in localization and aggregation of Joka2 in plant cells. We show that Joka2 can homodimerize not only through its N-terminal PB1-PB1 interactions but also via interaction between N-terminal PB1 and C-terminal UBA domains. We also demonstrate that Joka2 co-localizes with recombinant ubiquitin and sequestrates it into aggregates and that C-terminal part (containing UBA domains) is sufficient for this effect. Our results indicate that Joka2 accumulates in cytoplasmic aggregates and suggest that in addition to these multimeric forms it also exists in the nucleus and cytoplasm in a monomeric form.

Keywords: Joka2; NBR1; PB1; UBA; autophagy; proteasome; selective autophagy cargo receptor; ubiquitin.

Figure 1

Cytoplasmic and vacuolar localization of transiently expressed Joka2 and AtNBR1 in leaf epidermal cells of N. benthamiana. (A) BiFC assay of interaction between Joka2 and NtATG8f using randomly chosen combination (YN-NtATG8f+Joka2-YC) of the vectors. (B) Co-localization of co-expressed GFP-AtNBR1 and RFP-AtNBR1 (AtNBR1 fused to two variants of fluorescent protein). (C) Localization of Joka2-RFP in the vacuole. (D) Subcellular localization of co-expressed RFP-Joka2 and γ-TIP-CFP—a tonoplast marker based on an aquaporin of the vacuolar membrane fused to CFP. The enlarged part of the picture visualizes tonoplast (the blue fluorescence signal) of the central vacuole which surrounds the nucleus (N) and red fluorescence of RFP-Joka2 fusion protein observed mainly inside the vacuole close to the tonoplast as a smear (arrowheads) or in spots (arrows). Scale bar, 10 μm.

Figure 2

Nuclear localization of Joka2. (A) Subcellular localization of transiently expressed truncated Joka2 proteins (INT1-YFP, INT2-YFP, and PB1-YFP) in leaf epidermal cells of N. benthamiana treated (+LMB) and not treated (−LMB) with the inhibitor of nuclear export. The localization of INT1-YFP and the INT2-YFP was unaffected by the LMB treatment, while PB1-YFP remained in the nucleus only after treatment with LMB. White lines with arrows indicate the cross-section of the cells used in analysis shown to the left. The nuclei are stained blue with DAPI. (B) An rhizodermis cell of transgenic tobacco line J4-1 expressing Joka2-YFP (yellow) and DAPI staining (blue) indicating the nuclear localization of Joka2-YFP. Scale bar, 10 μm.

Figure 3

Truncated Joka2 containing only UBA domains co-localizes with ubiquitin linked to YFP (Ub-VV-YFP). Truncated Joka2 proteins lacking PB1, PB1, and ZZ, ZZ, and UBA or UBA domains were transiently co-expressed in N. benthamiana leaves with unstable ubiquitin linked to YFP (Ub-VV-YFP). The overlapping fluorescent signals were observed only in the case of co-expression of Ub-VV-YFP with the following versions of the recombinant proteins: full-length Joka2-CFP, ZZUBA-CFP, CFP-ZZUBA, UBA-CFP. Scale bar, 10 μm.

Figure 4

PB1 sequence analysis. (A) Alignment of PB1 domain sequences from tobacco Joka2, Arabidopsis NBR1, and Homo sapiens p62 and NBR1. Blue background color denotes basic residues and red background color denotes acidic residues from OPCA-motif important for PB1 domain interactions and self-interaction. (B) Alignment of PB1 domain sequences of Joka2, AtNBR1, and ubiquitin sequence from Arabidopsis thaliana. Green background color denotes similar residues between ubiquitin and PB1 domain of Joka2 and AtNBR1. Identical amino acids are indicated with asterisks and by dots are marked amino acids with high similarity. (C) PB1 domain from Joka2 modeled using Swissmodel (PBD: 2KKC). By blue color are marked basic residues and by red are colored acidic residues from OPCA-motif. Two surfaces are shown. (D) PB1 domain from Joka2 modeled using Swissmodel (PBD: 2KKC). Green color marks amino acids similar between ubiquitin and PB1 domain of Joka2. Two surfaces are shown.

Figure 5

BiFC assay of dimerization of transiently expressed Joka2 and AtNBR1 in leaf epidermal cells of N. benthamiana. Four combinations of BiFC plasmids were used for analysis of Joka2 dimerization (YC-Joka2+YN-Joka2, YC-Joka2+Joka2-YN, Joka2-YC+Joka2-YN, and Joka2-YC+YN-Joka2). Joka2-Joka2 interaction (green signal) was observed in all combinations. Dimerization of AtNBR1 was tested by BiFC method using only one randomly chosen plasmids combination (YC-AtNBR1+YN-AtNBR1). Scale bar, 10 μm. Negative controls are shown in Figure S5.

Figure 6

BiFC assay of dimerization of PB1 in planta. The combinations of plasmids (PB1-YC+YN-PB1, PB1-YC+PB1-YN, YC-PB1+PB1-YN, YC-PB1+YN-PB1, and PB1ZZ-YC+PB1ZZ-YN) were used for BiFC analysis in leaf epidermal cells of N. benthamiana. The interaction (green signal) was mainly observed in cytosolic aggregates. For the combination of PB1-YC+YN-PB1 no fluorescence signal was observed in plant cells. For the combination of YC-PB1+YN-PB1 the weak fluorescence in cytoplasm was also present. Two independent representative pictures are shown for the combination of YC-PB1ZZ+YN-PB1ZZ. Scale bar, 10 μm.

Figure 7

Involvement of PB1 and UBA domains in formation of Joka2-Joka2 aggregates in planta. (A) Localization of truncated forms of Joka2 in N. benthamiana epidermal cells. (B) Joka2 subcellular localization analysis after co-expression of Joka2-YFP with various truncated forms of Joka2 linked to CFP in N. benthamiana leaves. Scale bar, 10 μm.

Figure 8

Interaction between PB1 and UBA domains. (A) Yeast two-hybrid (Y2H) analysis demonstrating weak interaction between PB1 domain and the fragment containing UBA domains of Joka2. Truncated proteins lacking either PB1 or UBA domains were fused to AD or BD domain of GAL4 protein and co-expressed in yeast cells (AH109 strain). Positive and negative controls for protein interaction analysis were provided by Invitrogen. (B) BiFC assay for PB1-UBA interaction in planta. Indicated combinations of plasmids (PB1-YN+UBA-YC, YN-PB1+UBA-YC, YC-PB1+UBA-YN, PB1-YC+UBA-YN) were used for analysis. Scale bar, 10 μm.

Figure 9

Model explaining possible PB1-UBA interactions detected in this work. Involvement of Joka2 in targeting of the ubiquitinated proteins into the cytoplasmic or nuclear Ubiquitin-Proteasomal System (UPS) is only hypothetical.