Dissecting the mechanism of Ca2+-triggered membrane fusion: probing protein function using thiol reactivity.
1. Ca(2+)-triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage-specific native preparation fully enabling the directly coupled functional-molecular analyses necessary to identify critical components of fast triggered membrane fusion. 2. Recent work on lipidic components has established a direct role for cholesterol in the fusion mechanism via local contribution of negative curvature to readily enable the formation of transient lipidic fusion intermediates. In addition, cholesterol- and sphingomyelin-enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca(2+) transient. 3. There is less known about the identity of proteins involved in the Ca(2+)-triggering steps of membrane fusion. Thiol reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol-reactive reagents have identified different effects on Ca(2+) sensitivity and the extent of fusion, suggesting that there are at least two distinct thiol sites that participate in the fusion mechanism: one that regulates the efficiency of Ca(2+) sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca(2+) sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was used to potentiate fusion and simultaneously tag the proteins involved. Ongoing work involves the isolation of cholesterol-enriched membrane fractions to reduce the complexity of the labelled proteome, narrowing the number of candidate proteins.
Furber
Kendra L
KL
Department of Physiology and Pharmacology, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
Dean
Kwin T
KT
Coorssen
Jens R
JR
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D007546:Isoquinolines / Q000737:chemistry; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D012617:Sea Urchins; D022142:Secretory Vesicles / Q000737:chemistry / Q000502:physiology; D013109:Sphingomyelins / Q000737:chemistry / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D007546:Isoquinolines; D008565:Membrane Proteins; D013109:Sphingomyelins; D013438:Sulfhydryl Compounds; C017475:lucifer yellow; D002784:Cholesterol; D002118:Calcium