Assembly-hub function of ER-localized SNARE proteins in biogenesis of tombusvirus replication compartment
(A) Top panel: The split ubiquitin assay was used to test binding between the TBSV p33 replication protein and Ufe1p in yeast. The bait p33 was co-expressed with N-terminally-tagged Ufe1p protein. SSA1 (HSP70 chaperone), and the empty prey vector (NubG) were used as positive and negative controls, respectively. Second-to-fifth panels: The split ubiquitin assay was used to test binding between p33 and AtSyp81 (the plant ortholog of the yeast Ufe1p), Use1p, Dsl1 and Sec20p, respectively, in yeast. Note that Dsl1p protein was C-terminally-tagged. (B) The split ubiquitin assay was used to test binding between the CIRV p36 replication protein and Ufe1p in yeast. See further details in panel A. (C) Co-purification of Ufe1p and Use1p with the p33 replication protein from subcellular membranes. Top panels: Western blot analysis of co-purified HA-tagged cellular Ufe1p protein (lanes 1–2) or Use1p (lanes 3–4) with Flag-affinity purified FLAG/His6-p33. Ufe1p and Use1p were detected with anti-HA antibody, while FLAG/His6-p33 was detected with anti-FLAG antibody (third and fourth panels). The negative control was His6-tagged p33 purified from yeast extracts using a FLAG-affinity column. Second panel: Western blot of total HA-Ufe1p or HA-Use1p in the total yeast extract using anti-HA antibody. Bottom panel: Western blot of total FLAG/His6-tagged p33 and His6-p33 in the total yeast extract using anti-His antibody.