Dissecting the mechanism of Ca2+-triggered membrane fusion: Probing protein function using thiol reactivity

• Published in: Clinical and experimental pharmacology & physiology 2010-02, Vol.37 (2), p.208-217
• Main Authors: Furber, Kendra L, Dean, Kwin T, Coorssen, Jens R
• Format: Article
• Language: English
• Subjects: Animals; calcium; Calcium - metabolism; Cholesterol - chemistry; Cholesterol - metabolism; cortical vesicles; Isoquinolines - chemistry; Membrane Fusion; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Protein Interaction Domains and Motifs; protein thiols; Sea Urchins; Secretory Vesicles - chemistry; Secretory Vesicles - physiology; Sphingomyelins - chemistry; Sphingomyelins - physiology; Sulfhydryl Compounds - chemistry; Sulfhydryl Compounds - metabolism; two-dimensional gel electrophoresis
• ISSN: 0305-1870; 1440-1681
• DOI: 10.1111/j.1440-1681.2009.05278.x

Summary 1. Ca2+‐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 Ca2+ transient. 3. There is less known about the identity of proteins involved in the Ca2+‐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 Ca2+ 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 Ca2+ sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca2+ 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.