Cell Adhesion Molecule DdCAD-1 Is Imported into Contractile Vacuoles by Membrane Invagination in a Ca2+- and Conformation-dependent Manner

Shrivani Sriskanthadevan,Teresa Lee,Zhi Lin,Daiwen Yang,Chi-Hung Siu

doi:10.1074/jbc.m109.057257

Shrivani Sriskanthadevan, Teresa Lee + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.057257

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Abstract

The cadA gene in Dictyostelium encodes a Ca(2+)-dependent cell adhesion molecule DdCAD-1 that contains two beta-sandwich domains. DdCAD-1 is synthesized in the cytoplasm as a soluble protein and then transported by contractile vacuoles to the plasma membrane for surface presentation or secretion. DdCAD-1-green fluorescent protein (GFP) fusion protein was expressed in cadA-null cells for further investigation of this unconventional protein transport pathway. Both morphological and biochemical characterizations showed that DdCAD-1-GFP was imported into contractile vacuoles. Time-lapse microscopy of transfectants revealed the transient appearance of DdCAD-1-GFP-filled vesicular structures in the lumen of contractile vacuoles, suggesting that DdCAD-1 could be imported by invagination of contractile vacuole membrane. To assess the structural requirements in this transport process, the N-terminal and C-terminal domains of DdCAD-1 were expressed separately in cells as GFP fusion proteins. Both fusion proteins failed to enter the contractile vacuole, suggesting that the integrity of DdCAD-1 is required for import. Such a requirement was also observed in in vitro reconstitution assays using His(6)-tagged fusion proteins and purified contractile vacuoles. Import of DdCAD-1 was compromised when two of its three Ca(2+)-binding sites were mutated, indicating a role for Ca(2+) in the import process. Spectral analysis showed that mutations in the Ca(2+)-binding sites resulted in subtle conformational changes. Indeed, proteins with altered conformation failed to enter the contractile vacuole, suggesting that the import signal is somehow integrated in the three-dimensional structure of DdCAD-1.

Highlights

In eukaryotes, soluble secretory proteins are typically transported through the classical ER3-Golgi transport pathway [1, 2]
We investigated the structural requirements for the import of DdCAD-1 into contractile vacuoles
Samples were subjected to SDS-PAGE cells were transfected with a DdCAD-1-green fluorescent protein (GFP) construct

Summary

EXPERIMENTAL PROCEDURES

Construction of DdCAD-1-GFP, N-GFP, and C-GFP Expression Vectors and Cell Transfection—For DdCAD-1-GFP expression, a 645-bp fragment of DdCAD-1 cDNA was cloned into the HindIII and EcoRI sites of pA15/NIGFP expression vector (kindly provided by Dr David Knecht, University of Connecticut). Proteolytic digestion of contractile vacuoles was carried out by incubating samples (containing ϳ100 ␮g of protein) with 0.01 mg/ml proteinase K in the presence or absence of 0.05% SDS at 37 °C for 1 h. In Vitro Reconstitution of DdCAD-1 Import into Contractile Vacuoles—Contractile vacuoles and cytosol fractions derived from cadA-null cells were mixed at a 1:1 ratio in terms of protein amount in a 600-␮l reaction sample to give a final concentration of 2 mg/ml. The contractile vacuoles were pelleted at 12,000 rpm for 10 min at 4 °C, washed twice with TM buffer, and subjected to proteinase K (10 ␮g/ml) digestion for 1 h at 37 °C in the presence or. Grown cadA null transfectants (2 ϫ 107 cells/ml) were developed in 17 mM phosphate buffer for 6 h at room temperature. The cross-linked His6-DdCAD-1 was subjected to the contractile vacuole import assay

RESULTS

In Vitro Import Analysis of GFP

DISCUSSION

To further investigate the structural mechanisms involved in