A Novel Protein LZTFL1 Regulates Ciliary Trafficking of the BBSome and Smoothened
Figure 1
Identification of LZTFL1 as a BBSome interacting protein.
(A) Co-purification of Lztfl1 as a BBSome-interacting protein from LAP-BBS4 transgenic mouse testis. Lysates from WT and Tg testes were subjected to TAP and purified proteins were separated by SDS-PAGE and silver-stained. Protein size markers (M) were loaded in the left lane. (B) FS-LZTFL1 (blue arrowhead) and its associated proteins were isolated by TAP from HEK293T cells and visualized by silver staining. Parental cells were used as a control. Red arrowhead indicates endogenous LZTFL1, and asterisk, two smaller forms of LZTFL1. (C) LZTFL1 interacts with BBS9 within the BBSome. Each subunit of the BBSome (HA-tagged) was co-transfected with Myc-tagged LZTFL1 into HEK293T cells and lysates were analyzed by co-immunoprecipitation (IP) using anti-HA antibodies. Bottom panel shows immunoprecipitated BBS proteins and middle, Myc-LZTFL1 in the lysates. The top panel shows Myc-LZTFL1 precipitated by anti-HA antibodies. (D) BBS9 binds to the C-terminal half of LZTFL1. Full-length and several deletion mutant variants of Myc-LZTFL1 were co-transfected with HA-BBS9 in HEK293T cells. Myc-GFP was used as a negative control and anti-Myc antibody was used for IP. Numbers indicate expressed portions of LZTFL1 in amino acid positions. (E) The vast majority of Lztfl1 is not associated with the BBSome. Lysates from WT mouse testis and eye were subjected to size exclusion chromatography using Superose-6 10/300 GL column. Eluted fractions were analyzed by SDS-PAGE followed by immunoblotting against indicated antibodies. Elution volume of protein standards is shown at the bottom.