Sphingolipids modulate docking, Ca2+ sensitivity and membrane fusion of native cortical vesicles

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion

“…This Rab family 1 motif is largely conserved (>50%) among the Rab GTPases and across different species (Table 2), and this effector domain peptide has been previously shown to interfere with the formation of the fertilization envelope in urchin oocytes [47]. An addition of exogenous RAB peptide inhibited the extent of fusion by~63% in the modified settle assay, compared to~46% in the standard assay; this strongly indicates an effect on upstream stages of CV tethering/docking/priming [1][2][3]6,8,30,39]. With the addition of the RAB effector domain, there was no effect on Ca 2+ -sensitivity; however, after pretreatment with IA, treatment with the RAB peptide resulted in a decrease in Ca 2+ -sensitivity, mitigating the potentiation effect of IA.…”

“…Membrane fragments were isolated by centrifugation at 120,000× g. Cholesterol-enriched membrane fragments were further fractionated by density centrifugation on sucrose gradients (40%/30%/22%/10% sucrose, 25 mM Tris; pH 7), collected from the 22%/10% interface, washed in 25 mM Tris buffer and then isolated by centrifugation at 120,000× g. Membrane pellets were solubilized in 2DE sample buffer (8 M urea, 2 M thiourea, 4% CHAPS), supplemented with the broad-spectrum protease inhibitor cocktail, and protein concentration was determined using the EZQ Protein Quantitation Kit (ThermoFisher). Membrane proteins were resolved by well-established, optimized 2DE protocols, with slight modifications [2,8,40,50]. In the first dimension, proteins were resolved on immobilized nonlinear pH 3-10 gradients (3-10 NL IPG; Bio-Rad).…”

“…To further narrow the number of candidate proteoforms of interest, the isolation of cholesterol-enriched membrane fragments was used as a prefractionation tool. Cholesterol-and sphingomyelin-enriched domains regulate the efficiency of exocytosis by organizing critical components near the fusion site [8][9][10][11]45,46]. To this end, cholesterol was used as 'bait' to enrich proteoforms involved in regulated exocytosis, both to reduce the complexity of the proteome and improve the detection of low abundance labeled proteoforms [40,45].…”

“…This involves the coordinated actions of both lipid and protein components of the secretory vesicle membrane, particularly the later steps of Ca 2+ -triggered exocytosis. There have been recent advances in understanding how the lipid matrix may modulate upstream docking/priming steps [1][2][3][4][5][6][7], regulate the Ca 2+ sensitivity and efficiency of fusion [5][6][7][8][9][10][11], and facilitate or impede the merger of biological membranes [1,6,7,9,[12][13][14]. However, there remains debate regarding the identity of proteins involved in the Ca 2+ -sensing and triggering steps that regulate the efficiency of exocytosis.…”

“…As a well-established top-down approach, labeled proteoforms were analyzed by high-resolution, two-dimensional gel electrophoresis (2DE) followed by mass spectrometric analysis of tryptic spot digests for protein identification (i.e., MALDI-TOF/TOF or LC/MS/MS) [41][42][43][44]. Considering the evidence for lipid microdomain involvement in the exocytotic mechanism [8][9][10][11]45,46], labeled proteoforms from both total CV membrane and cholesterol-enriched CV membrane isolates were resolved by 2DE. Protein spots labeled by all three reagents, and detected in cholesterol-enriched membranes, included several Rab GTPases.…”