Regulated secretion: SNARE density, vesicle fusion and calcium dependence.
SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+ dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion (Ca2+ sensitivity and the catalysis and execution of fusion) require additional proteins that function downstream of SNAREs.
Coorssen
Jens R
JR
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA.
Blank
Paul S
PS
Albertorio
Fernando
F
Bezrukov
Ludmila
L
Kolosova
Irina
I
Chen
Xiongfong
X
Backlund
Peter S
PS
Zimmerberg
Joshua
J
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D015151:Immunoblotting; D007700:Kinetics; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D012617:Sea Urchins; D050825:Synaptosomal-Associated Protein 25; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D014357:Trypsin; D002118:Calcium