The priming factor CAPS1 regulates dense-core vesicle acidification by interacting with rabconnectin3β/WDR7 in neuroendocrine cells

Ellen Crummy,Muralidharan Mani,John C Thellman,Thomas F.J Martin

doi:10.1074/jbc.ra119.007504

Ellen Crummy, Muralidharan Mani + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra119.007504

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Abstract

Vacuolar-type H+-ATPases (V-ATPases) contribute to pH regulation and play key roles in secretory and endocytic pathways. Dense-core vesicles (DCVs) in neuroendocrine cells are maintained at an acidic pH, which is part of the electrochemical driving force for neurotransmitter loading and is required for hormonal propeptide processing. Genetic loss of CAPS1 (aka calcium-dependent activator protein for secretion, CADPS), a vesicle-bound priming factor required for DCV exocytosis, dissipates the pH gradient across DCV membranes and reduces neurotransmitter loading. However, the basis for CAPS1 binding to DCVs and for its regulation of vesicle pH has not been determined. Here, MS analysis of CAPS1 immunoprecipitates from brain membrane fractions revealed that CAPS1 associates with a rabconnectin3 (Rbcn3) complex comprising Dmx-like 2 (DMXL2) and WD repeat domain 7 (WDR7) proteins. Using immunofluorescence microscopy, we found that Rbcn3α/DMXL2 and Rbcn3β/WDR7 colocalize with CAPS1 on DCVs in human neuroendocrine (BON) cells. The shRNA-mediated knockdown of Rbcn3β/WDR7 redistributed CAPS1 from DCVs to the cytosol, indicating that Rbcn3β/WDR7 is essential for optimal DCV localization of CAPS1. Moreover, cell-free experiments revealed direct binding of CAPS1 to Rbcn3β/WDR7, and cell assays indicated that Rbcn3β/WDR7 recruits soluble CAPS1 to membranes. As anticipated by the reported association of Rbcn3 with V-ATPase, we found that knocking down CAPS1, Rbcn3α, or Rbcn3β in neuroendocrine cells impaired rates of DCV reacidification. These findings reveal a basis for CAPS1 binding to DCVs and for CAPS1 regulation of V-ATPase activity via Rbcn3β/WDR7 interactions.

Highlights

Vacuolar-type H؉-ATPases (V-ATPases) contribute to pH regulation and play key roles in secretory and endocytic pathways
CAPS1 is a Dense-core vesicles (DCVs)-resident protein with a key role in vesicle docking and priming and in sensitive factor–associated protein receptor (SNARE) complex formation for DCV fusion with the plasma membrane [40, 45]
The knockdown of CAPS1 in hippocampal neurons increases the pH of DCVs from 5.8 to 6.7 [50], which is likely responsible for other knockdown phenotypes such as impaired catecholamine loading [46, 47, 63] and disruption of chromogranin packaging [3, 49]

Summary

The abbreviations used are

V-ATPase, vacuolar-type Hϩ-ATPase; DCV, dense-core vesicle; CAPS, Ca2ϩ-dependent activator protein for secretion; RAVE, regulator of V-ATPase of vacuoles and endosomes; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; PH, pleckstrin homology; Rbcn, rabconnectin; MHD, Munc homology domain; from the endoplasmic reticulum to Golgi to post-Golgi vesicles and from early to late endosomes [1, 2]. DUF1041, domain of unknown function; VMAT, vesicular monoamine transporter; DMXL2, Dmx-like 2; WDR7, WD repeat domain 7; SNARE, soluble N-ethylmaleimide–sensitive factor–associated protein receptor; BDNF, brain-derived neurotrophic factor; NPY, neuropeptide Y; EGFP, enhanced GFP; NT, nontargeting; HEK, human embryonic kidney; HA, hemagglutinin; TIRF, total internal reflection fluorescence; GAPDH, glyceraldehyde-3phosphate dehydrogenase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GaAsP, gallium arsenide phosphide; NA, numerical aperture; NP-40, Nonidet P-40; ROUT, robust regression and outlier removal. These studies reveal part of the basis for CAPS1 binding to DCVs and for CAPS1 regulation of V-ATPase activity in concert with Rbcn3␤/WDR7

Results

Discussion

Experimental procedures