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. 2016 May 9;213(3):305-14.
doi: 10.1083/jcb.201601089. Epub 2016 May 2.

Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network

Alvaro H Crevenna  1 , Birgit Blank  2 , Andreas Maiser  3 , Derya Emin  4 , Jens Prescher  4 , Gisela Beck  2 , Christine Kienzle  2 , Kira Bartnik  4 , Bianca Habermann  2 , Mehrshad Pakdel  2 , Heinrich Leonhardt  3 , Don C Lamb  4 , Julia von Blume  5
Affiliations

Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network

Alvaro H Crevenna et al. J Cell Biol. .

Abstract

Sorting and export of transmembrane cargoes and lysosomal hydrolases at the trans-Golgi network (TGN) are well understood. However, elucidation of the mechanism by which secretory cargoes are segregated for their release into the extracellular space remains a challenge. We have previously demonstrated that, in a reaction that requires Ca(2+), the soluble TGN-resident protein Cab45 is necessary for the sorting of secretory cargoes at the TGN. Here, we report that Cab45 reversibly assembles into oligomers in the presence of Ca(2+) These Cab45 oligomers specifically bind secretory proteins, such as COMP and LyzC, in a Ca(2+)-dependent manner in vitro. In intact cells, mutation of the Ca(2+)-binding sites in Cab45 impairs oligomerization, as well as COMP and LyzC sorting. Superresolution microscopy revealed that Cab45 colocalizes with secretory proteins and the TGN Ca(2+) pump (SPCA1) in specific TGN microdomains. These findings reveal that Ca(2+)-dependent changes in Cab45 mediate sorting of specific cargo molecules at the TGN.

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Figures

Figure 1.
Figure 1.
Cab45 self-assembles in the presence of Ca2+. (A) Purified Golgi membranes from HeLa cells were incubated with DMSO (control) or 25 µM BAPTA-AM for 15 min at 37°C. Subsequently, membranes were lysed in NativePAGE buffer containing 1% DDM, subjected to NativePAGE (3–12%, top) or denaturing SDS-PAGE and analyzed by WB with TGN46 (middle) or anti-Cab45 (bottom) antibodies. (B) Top: HeLa cells were fixed and stained with a Cab45 and an Alexa Fluor 488–labeled antibody and analyzed by laser scanning confocal microscopy (LSCM). Bottom two panels: HeLa cells were incubated with a Ca2+-containing buffer supplemented with DMSO (control) or a Ca2+-free Hanks’ buffer containing 2 µM ionomycin and 5 mM EGTA (Palmer and Tsien, 2006) for 10 min. Then, cells were fixed and stained with the Cab45 antibody and a secondary, Cy5-labeled antibody. dSTORM imaging was performed using wide-field illumination and single fluorescent emitters were localized using a Gaussian least-squares fit. Bars, 10 µm. (C) Recombinant Cab45 was incubated in calcium-free buffer, with 1 mM Ca2+ or with 1 mM Ca2+ and 2 mM EGTA. Samples were subjected to NativePAGE (top) or SDS-PAGE (bottom) and analyzed by WB with an anti-Cab45 antibody. (D) Recombinant ATTO-488–labeled Cab45 was incubated as in (C) and analyzed by confocal microscopy. (E) Recombinant ATTO-488-labeled Cab45 was treated with 10 or 100 mM Mg2+ and analyzed by confocal microscopy. Bars, 20 µm (F) Oligomerization of Cab45 was assessed by fluorescence microscopy as a function of Ca2+ and protein concentration. (G) Far-UV CD analysis of recombinant Cab45 in the presence and absence of Ca2+. (Inset) Ellipticity of Cab45 at 222 nm plotted as a function of Ca2+ concentration.
Figure 2.
Figure 2.
Cargo recruitment by Cab45 oligomers. (A–C) Pull-down experiments performed with His-tagged Cab45 and the potential cargo molecules LyzC (A), COMP (B), and CathepsinD (C), analyzed by SDS-PAGE and Coomassie staining. (D–F) In vitro oligomerization assay. Cab45 and LyzC (D), COMP (E) or CathepsinD (F), labeled as indicated, were incubated in Ca2+-free buffer, with Ca2+ or Ca2+/EGTA. Bars, 20 µm. (G) Pearson coefficient of colocalization between Cab45-ATTO488 and potential cargo molecules. Error bars represent SD from three independent experiments (n = 3). Two images were quantified per experiment. (H and I) FRAP analysis of LyzC-Cy5 (H) and COMP–Alexa Fluor 647 (I) recruited to individual unlabeled Cab45 oligomers. The black line is the mean signal, whereas the light blue trace represents one SD on either side (n = 13 for LyzC and 15 for COMP). The insets show examples of a Cab45 oligomer before bleaching (prebleach), immediately after bleaching (bleach), and ∼10 min after bleaching (590 s). Bar, 2 µm.
Figure 3.
Figure 3.
Cab45 Ca2+ binding is required oligomerization in living cells. (A) Cab45−/− cells were generated using CRISPR/Cas9 with 2 gRNAs targeting the SDF4 (Cab45) locus as described in Materials and methods. Single clones were picked and analyzed by WB with a Cab45 antibody. (B) Structural model of human Cab45. The three EFh pairs are highlighted in blue, and the glutamates (E) in contact with the ligand are shown in orange (EFh 1), red (EFh 2), and green (EFh 3). The Ca2+-binding–deficient mutant (Cab45-HA 6EQ-mut), in which all six Glu residues have been replaced by Gln, was described previously (von Blume et al., 2012). (C) Cells of the Cab45−/− knockout line, and its stable transfectants Cab45−/− Cab45-HA WT or Cab45−/− Cab45-HA 6EQ-mut, were fixed and stained with Cab45 (green), SPCA1 (red), and TGN46 (blue) antibodies and analyzed by fluorescence microscopy. Bars, 5 µm. (D) Cab45−/− cells expressing either Cab45-HA WT or Cab45-HA 6EQ-mut were incubated at 20°C in the presence of cycloheximide to allow proteins to accumulate in the TGN. After 2 h, cells were shifted to 37°C for an additional 2 h, and cells and media were collected and analyzed by Western blotting with the Cab45 antibody. (E) Golgi membranes extracted from Cab45−/−, Cab45−/− Cab45-HA WT, or Cab45−/− Cab45-HA 6EQ-mut lines were lysed in NativePAGE buffer containing 1% DDM, subjected to NativePAGE (3–12%) or SDS-PAGE, and analyzed by WB with a Cab45 or anti-TGN46 antibody.
Figure 4.
Figure 4.
Cab45 Ca2+ binding is required for sorting in living cells. (A) Cell lysates of Cab45−/−, Cab45−/− Cab45-HA WT or Cab45−/− Cab45-HA 6EQ-mut HeLa cells were analyzed by WB with a Cab45 antibody. (B and D) Cell culture supernatants of the same cells were incubated in serum-free medium for 4 h at 37°C. Media and cell extracts were blotted and probed with antibodies against COMP (B) or Flag (LyzC; D). Western blots from three independent experiments were quantified by densitometry using the ImageJ software. Bar graphs represent the densitometry values of external COMP (C) and LyzC (E) normalized to internal protein contents, respectively. Error bars represent SD calculated of three independent experiments of secreted COMP (C) or LyzC (E), respectively (n = 3). **, P < 0.01 (compared datasets).
Figure 5.
Figure 5.
SPCA1, Cab45 and cargo cluster in the TGN. To obtain higher resolution, 3D SIM was applied. HeLa cells expressing HA-SPCA1 were fixed and stained with anti-HA and anti-Cab45 antibodies and phalloidin and analyzed by 3D SIM. (A) Representative cross section showing the locally overlapping distributions of SPCA1 and Cab45 in the TGN. Bar, 5 µm. (B) HeLa cells expressing Flag-LyzC were fixed and stained with Flag (LyzC) and Cab45 antibodies. Bar, 5 µm. (C) HeLa cells were fixed and stained with p230 and Cab45 antibodies. Bar, 5 µm. (D) HeLa cells were fixed and stained with TGN46 and Cab45 antibodies. (E) Probability (i.e., normalized frequency) of Cab45 colocalization with SPCA1, LyzC, p230, and TGN46. Depiction of the image quantification is shown in Fig. S3 (C, F, I, and L). (F) Schematic depiction of the putative Cab45-based sorting mechanism. Cab45 undergoes a conformational change upon the SPCA1-mediated local increase in Ca2+ concentration in the TGN lumen. As a consequence, Cab45 binds cargo proteins and separates them from other soluble proteins in the TGN by sequestering them in putative subdomains. This sorting mechanism might enable cells to pack soluble cargos into vesicles and recycle Cab45 for a new cycle of sorting by extrusion of internal Ca2+.
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