Figure 4: LPAATδ is required for the fission of basolaterally-directed carriers.
(a,b) Representative images of COS7 cells treated with non-targeting and LPAATδ siRNAs in a or LPAATγ siRNAs in b before VSV infection and the TGN-exit assay with 0.5% tannic acid. The cells were fixed following a 20 °C block (0 min) or 30 min after the shift to 32 °C, and stained for VSVG-positive post-Golgi carriers. Quantification is on the right (see Methods). (c–e) Representative images of COS7 cells treated with non-targeting and LPAATδ siRNAs before co-transfection for the last 16 h with VSVG-ts045-KDELR-myc in c or with the endocytosis-defective LDLR–GFP receptor in d or with a plasmid encoding p75–GFP in e. (c) The distribution of the chimeric KDELR was examined following the shift to the non-permissive temperature for 30 min. Quantification of ER distribution of the chimeric KDELR (right). (d) Following the 2 h at 20 °C transport block (0 min) and 60 min after the shift to 32 °C (with cycloheximide), the cells were fixed and labelled with TGN46 (Golgi marker; red). Insets: enlarged view of merged signals for the Golgi area. (e) Following the 3 h at 20 °C block (0 min) and 60 min after the shift to 32 °C (with cycloheximide), the cells were fixed and stained for GM130 (Golgi marker; red). (d,e) Quantification of LDLRY18A–GFP in d and p75–GFP in e in the Golgi area (right). (f) Representative microscopy of HeLa cells stably transfected with hGH-FM–GFP and treated with non-targeting and LPAATδ siRNAs before subjection to a secretion assay. Release of hGH-FM–GFP from ER was performed by the addition of DD-solubilizer at 37 °C for the indicated times. Quantification of hGH-FM–GFP in the Golgi area (right). (a–e), Dotted lines indicate cell borders. (a–f), The efficiency of interference was monitored by western blotting of the cell lysates using polyclonal anti-LPAATδ in a,c–f, or polyclonal anti-LPAATγ in b antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown for the internal protein levels and molecular weight standards (kDa) are indicated on the left of each panel in a–f. Data are means±s.d. of three independent experiments. ***P<0.005 (Student’s t-tests). Scale bars, 10 μm.
