AtTPR7 as part of the Arabidopsis Sec post-translocon

Abstract

The secretory system in eukaryotic organisms ensures targeting of proteins to their place of function after they entered the endoplasmic reticulum either co- or post-translationally. Thereby proteins are translocated through the Sec translocon into the endoplasmic reticulum. In the Arabidopsis genome homologs for the three major components of the Sec translocon, the central pore Sec61α and the auxiliary proteins Sec62 and Sec63 are present. Phylogenetic analyses show Sec61α to be the most conserved subunit within the Sec translocon whereas Sec62 and Sec63 show less homology but contain the same functional domains among all organisms. We recently characterized a novel tetratricopeptide repeat domain containing protein, AtTPR7, as part of the Arabidopsis Sec translocon which is probably involved in chaperone assisted post-translational import. In this study we investigated the interaction of AtTPR7 with Sec62 as well as the cytosolic chaperones HSP70 and HSP90 not only in vitro but also in vivo to further strengthen the hypothesis of AtTPR7 being a chaperone docking protein of the Sec translocon for secretory preproteins in Arabidopsis.

Keywords: Arabidopsis thaliana; AtTPR7; HSP70; HSP90; Sec translocon; endoplasmic reticulum; post-translational import.

Figure 1. Domain structure of Sec62 and Sec63 in mammals, plants and yeast. (A) Comparison of the Sec62 protein of Homo sapiens, NP_003253.1; Mus musculus, NP_081292.1; Arabidopsis thaliana, At3g20920; Oryza sativa, Os02 g0435000; Saccharomyces cereviseae , NP_015231.2. (B) Comparison of the Sec63 protein of Homo sapiens, NP_009145.1; Mus musculus, NP_694695.3; Arabidopsis thaliana, At1g79940, At4g21180; Oryza sativa, Os04 g0307200; Saccharomyces cereviseae, NP_014897.1. Specific domains are indicated in colors. Predicted transmembrane domains are indicated as gray bars. Homolog genes were identified by “HomoloGene” (NCBI), conserved domains by “Conserved Domain Search” (NCBI) and transmembrane domains were predicted with the help of TMHMM (CBS).

Figure 2. Interaction of AtTPR7 with Sec62.(A) Recombinant AtTPR7-His (20 µg) (At5g21990) was incubated for 1 h with 15 µl of radiolabeled Sec62 translation product in 300 µl 1×PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄; pH 7.3). AtTPR7-His was re-purified by incubation with 20 µl Ni-NTA sepharose for 1 h at RT. the Ni-NTA sepharose was washed for three times with 1 ml of 1× PBS buffer containing 50 mM imidazole and samples were subsequently eluted with 20 µl of 1× PBS containing 300 mM imidazole. Five percent of the translation (TL), 2% of the flow through (FT), 2% of the first washing step (W1) and the total eluates (E) were subjected on a 10% SDS-PAGE. Association of Sec62 was detected by autoradiography. A sample without AtTPR7-His constructs was used as a control. (B) For bimolecular fluorescence complementation experiments the N-terminal part of Venus was fused to the N-terminus of AtTPR7 and the C-terminal part of SCFP to the N-terminus of Sec62. The constructs were co-transformed with the ER mCherry marker (middle panel) and transiently expressed in tobacco leaves. As a control Venus^N-AtTPR7 was cotransformed with SCFP3A^C alone (empty vector) and the ER mCherry marker (bottom panels). Images of tobacco leaf cells were obtained by confocal laser scanning microscopy (Leica, Type: TCS SP5; objective lense: HCX PL APO CS; magnification: 63×; numerical aperture: 1.3; imaging medium: glycerol; software: Leica Application Suite/Advanced Fluorescence). Reconstituted fluorescence obtained by close proximity of the Venus and the SCFP parts was monitored at 515 nm (left panel). Overlay of the signal at 515 nm and the mCherry marker is shown (right panel). Scale bars: 10 µm.

Figure 3. Interaction of AtTPR7 with AtHSP70.1 and AtHSP90.2. For bimolecular fluorescence complementation experiments the N-terminal part of Venus was fused to the N-terminus of AtTPR7 and the C-terminal part of SCFP to the N-terminus of AtHSP70.1 (At5g02500) and AtHSP90.2 (At5g56030), respectively. The constructs were cotransformed with the ER mCherry marker (middle panel) and transiently expressed in tobacco leaves. As a control Venus^N-AtTPR7 was cotransformed with SCFP3A^C alone and the ER mCherry marker (bottom panels). Images of tobacco leaf cells were obtained by confocal laser scanning microscopy. Reconstituted fluorescence was monitored at 515 nm (left panel). Overlay of the signal at 515 nm and the mCherry marker is shown (right panel). Scale bars: 10 µm.