The critical role of Exo84p in the organization and polarized localization of the exocyst complex.
The exocyst complex plays an essential role in tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. However, how the exocyst complex is assembled and targeted to sites of secretion is unclear. Here, we have investigated the role of the exocyst component Exo84p in these processes. We have generated an array of temperature-sensitive yeast exo84 mutants. Electron microscopy and cargo protein traffic analyses of these mutants indicated that Exo84p is specifically involved in the post-Golgi stage of secretion. Using various yeast mutants, we systematically studied the localization of Exo84p and other exocyst proteins by fluorescence microscopy. We found that pre-Golgi traffic and polarized actin organization are required for Exo84p localization. However, none of the exocyst proteins controls Exo84p polarization. In addition, Sec3p is not responsible for the polarization of Exo84p or any other exocyst component to the daughter cell. On the other hand, several exocyst members, including Sec10p, Sec15p, and Exo70p, clearly require Exo84p for their polarization. Biochemical analyses of the exocyst composition indicated that the assembly of Sec10p, Sec15p, and Exo70p with the rest of the complex requires Exo84p. We propose that there are at least two distinct regulatory mechanisms for exocyst polarization, one for Sec3p and one for the other members, including Exo84p. Exo84p plays a critical role in both the assembly of the exocyst and its targeting to sites of secretion.
Zhang
Xiaoyu
X
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018, USA.
Zajac
Allison
A
Zhang
Jian
J
Wang
Puyue
P
Li
Ming
M
Murray
John
J
TerBush
Daniel
D
Guo
Wei
W
D043402:Cathepsin A / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D016764:Cell Polarity; D005089:Exocytosis / Q000502:physiology*; D006056:Golgi Apparatus; D049452:Green Fluorescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008854:Microscopy, Electron; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D022142:Secretory Vesicles / Q000502:physiology / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014162:Transfection
C120810:EXO84 protein, S cerevisiae; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D043402:Cathepsin A