Anti-SMT1 | Sterol methyltransferase 1

Protein structure and subcellular localization of PTRE1.(a) PTRE1 has conserved FP and proline-rich domains similar to human PI31, plant-specific domains and a N-terminal transmembrane region (upper panel). An amino acid alignment of the C-terminal proline-rich domain, which is conserved in the predicted proteasome inhibitor family of eukaryotic species (hPI31, Homo sapiens; LOC682071, Rattus norvegicus; and Os07g054890, Oryza sativa), is shown (bottom panel). The R(Ar)DP motif is indicated by the purple rectangle and the conserved amino acid residues of these four proteins are highlighted with different colours. (b) PTRE1 localizes at nucleus (upper; scale bar, 20??m), plasma membrane (middle; bar, 20??m) and ER (bottom; bar, 20??m). Pavement cells of Arabidopsis seedlings expressing PTRE1-GFP were stained with DAPI (2??g?ml?1) to show the nuclear localization of PTRE1 (upper, DAPI stains nucleus and plant cell walls). Hypocotyl cells of 7-day-old Arabidopsis seedlings expressing PTRE1-GFP and plasma membrane aquaporin PIP2-RFP was observed after plasmolysis (middle). Plasmolysis was performed by adding 0.6?M mannitol for 30?min. PTRE1-GFP was transiently expressed in N. benthamiana leaves with ER-mCherry (bottom) and observed. (c) Enlarged ER localization of PTRE1 by transient expression of PTRE1-GFP or ER-mCherry in N. benthamiana leaves. Scale bar, 20??m. (d) Parts of PTRE1 protein were localized and surface-exposed at the plasma membrane. Plant materials were treated with the membrane-impermeable sulpho-NHS-SS-biotin reagent (+). Surface-exposed protein eluted after purification using biotin beads. Plasma membrane protein H+-ATPase and ER membrane protein SMT1 were used as control. Equal amounts of samples were subjected to SDS–PAGE and immunoblot analysed using anti-PTRE1 (rabbit), anti-H+-ATPase (rabbit) or anti-SMT1 (rabbit) antibodies.