Fig. 3.

NEV localizes to the trans-Golgi network (TGN) and endosomes. (A) NEV antiserum recognizes a ∼55 kDa protein in wild-type flower extracts. As the nev-2 allele encodes a truncated protein lacking the C-terminal recognition sequence of the NEV antiserum, no protein is detected in nev-2 flower extracts. Ponceau Red staining was used as a protein-loading control. (B-O) Immunofluorescent localization of NEV and endomembrane markers in primary root epidermal cells of wild-type (B), nev (C) and transgenic marker (D-O) plants. In M-O, the primary roots of YFP-RabA1e plants were incubated for 1 hour with 100 μM BFA before fixation and immunofluorescent staining. (B,C) NEV antiserum detects NEV protein in punctate structures in wild-type cells (B). Background fluorescence is shown for nev-2 cells (C). (D-I) NEV localization is distinct from the Golgi (D-F), and shows more precise localization with YFP-VTI12, a marker of the TGN (arrows, G-I). Occasional spots labeled by NEV (arrowheads, G-I) do not co-localize with VTI12. (J-O) NEV also co-localizes with YFP-RabA1e, a novel endosomal marker proposed to localize to the recycling endosome (arrows, J-L). BFA treatment of the primary root causes both NEV and YFP-RabA1e to localize to BFA bodies (M-O). Whereas the localization of YFP-RabA1e is confined to the core of the BFA bodies, NEV is also found at the periphery (arrows, O). Individual channels: YFP markers, green; NEV, red. Scale bars: 5 μm.