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Fig. 7. Sec31B interacts with Sec13 and Sec23. (A) The distribution of full-length and truncated Sec31B was compared with endogenous Sec23 in COS-7 and MDCK cells. Fluorescent mages from the COS-7 cells are shown. In all panels, endogenous wt Sec23 is stained red; Sec31B (wild-type or transfected) is stained green. Areas of coincidence appear yellow. (Top and middle rows) Comparison of the endogenous proteins or with transfected eYFP-Sec31B-F; overlap is extensive. (Bottom) Comparison of wild-type Sec23 with transfected eYFP-Sec31B-T; notice the absence of overlap. (EM) Thin sections of MDCK-787 cells embedded in LR white were stained with colloidal gold for Sec23 (5 nm particles) and for eYFP-Sec31B-F (10 nm particles). Notice the close association of staining over regions of vesicular tubular clusters (vtc) and on some 50-60 nm vesicles indicated by *. There is no staining of the Golgi cisternae (G). (B) Comparison of the distribution of endogenous Sec13 and eYFP-Sec31B-F in MDCK-787 cells that were also transfected with VSV-G, five minutes after incubation at permissive temperature (32°C). Staining of VSV-G is not shown, it was coincident with Sec13. Notice the perfect association of Sec13 staining with VSV-G in the punctate ERESs. (IP) Immunoprecipitation of the MDCK-787 cells with antibodies against GFP (Sec31B) or Sec13 co-precipitate Sec13. Non-immune sera (ctrl); cell lysates (lys). (GST pull-down) The direct interaction between Sec31B and Sec13 is mediated by the WD-repeat domain of Sec31B because a GST-fusion peptide encompassing the WD region of Sec31B efficiently binds Sec13 in MDCK cell lysates (WD), whereas a similar fusion protein derived from the C-terminal proline-rich domain of Sec31B (PRD) that is devoid of the WD region does not bind Sec13. Ctrl, GST alone; lys, MDCK cell lysate used in the pull-down assay.
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