Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p.
Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.
Boyd
Charles
C
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA.
Hughes
Thom
T
Pypaert
Marc
M
Novick
Peter
P
D008841:Actin Cytoskeleton / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D022081:Cell Surface Extensions / Q000378:metabolism / Q000648:ultrastructure; D005089:Exocytosis / Q000502:physiology*; D036681:Fluorescence Recovery After Photobleaching; D007150:Immunohistochemistry; D046911:Macromolecular Substances / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D046529:Microscopy, Electron, Transmission; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins
C103132:EXO70 protein, S cerevisiae; D046911:Macromolecular Substances; C103130:SEC3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins