Figure 3. Ca²⁺ binding by Cab45 is required for sorting of a Cab45 client, lysozyme C (LyzC), into Golgi-derived vesicles.

(A) HeLa cells were transfected with Cab45 siRNA and a plasmid that directs expression of siRNA-insensitive HA-epitope tagged Cab45-WT, or the Ca²⁺ binding defective Cab45–6EQ mutant variant. All cells were co-transfected with plasmids that direct expression of a LyzC – streptavidin binding peptide (SBP) – eGFP fusion protein (LyzC-SBP-eGFP), and streptavidin-KDEL ‘anchor’ that confers ER retention of SBP-containing proteins. Biotin was added to the culture medium to elicit release of LyzC-SBP-eGFP from the ER (0 min). Micrographs were captured after fixing cells 20, 40, and 60 minutes after biotin addition and the number of cytoplasmic vesicles was determined at each time point. Arrowheads point to cytoplasmic vesicles. Note that Golgi derived vesicles containing LyzC-SBP-eGFP are abundant in the cytoplasm of cells that express native, but not Ca²⁺ binding defective, Cab45. Magenta arrowheads point to Cab45–6EQ cytoplasmic vesicles that localize to distinct vesicles from LyzC-SBP-eGFP. See also Fig S1. Vesicle counts from at least 22 cells per condition are plotted in (B). The means of 3 independent experiments (± s.d.) are plotted. (C) Cathepsin D and lysozyme C fusion proteins are exported from the Golgi in different vesicles. HeLa cells were transfected with plasmids that direct expression of LyzC-SBP-eGFP or SS-SBP-tagRFP-Cathepsin D fusion proteins. Proteins were released from the ER by the addition of biotin and the cargo loads of Golgi derived cytoplasmic vesicles was determined as described in the legend to panel A. Micrographs show representative cells 40 minutes after release of cargo from the ER. Bars, 5 μm. (D) Export of Cathepsin D from the Golgi is unaffected by Cab45 gene silencing. The SS-SBP-eGFP-Cathepsin D fusion protein was released from the ER of Cab45 siRNA-silenced, or control, cells by addition of biotin. The mean number of post-Golgi vesicles per cell (>39 cells per condition, ± s.d.) are plotted from 3 independent experiments as a function of time. Vesicle counts for Cab45 siRNA and control siRNA cell populations are not statistically significant. (E) Ca²⁺ binding by EFh modules 1 and 3 is required for efficient export of LyzC from the Golgi apparatus. Cells were depleted of endogenous Cab45 by siRNA and siRNA-insensitive Cab45 cDNAs with mutations in each of the EF hand modules were expressed. The number of cytoplasmic vesicles containing LyzC-SBP-eGFP per cell was determined after release of LyzC-SBP-eGFP from the ER. Only the data for EFh2 and EFh1+3 are shown in this figure; additional data is shown in Fig. S1. The mean vesicle counts from at least 38 cells per condition (± s.d.) are plotted.