The Role of Exo 70 in Epithelia Morphogenesis and Renal Tubulogenesis
Zusammenfassung der Projektergebnisse
The results described in this report were obtained by studying two different aspects of the exocyst complex and its function. In the first part, the N-terminal domain of Sec3, a member of the exocyst complex, was analyzed by crystallization and further described by functional genetics. In the second part, the molecular mechanisms involved in primary ciliogenesis regulation and their connection to the exocyst complex were studied. The exocyst is an evolutionarily conserved octameric complex involved in polarized exocytosis from yeast to humans. The Sec3 subunit of the exocyst acts as a spatial landmark for exocytosis through its ability to bind phospholipids and small GTPases. The structure of the N-terminal domain of Sec3 (Sec3N) was determined ab initio and defines a new subclass of pleckstrin homology (PH) domains along with a new family of proteins carrying this domain. Respectively, N- and C-terminal to the PH domain Sec3N presents an additional α-helix and two β-strands that mediate dimerization through domain swapping. The structure identifies residues responsible for phospholipid binding, which when mutated in cells impair the localization of exocyst components at the plasma membrane and lead to defects in exocytosis. Through its ability to bind the small GTPase Cdc42 and phospholipids, the PH domain of Sec3 functions as a coincidence detector at the plasma membrane. Recent studies have implicated the exocyst in primary cilium formation. Primary cilia are microtubule-based membrane projections located at the surface of many cells. Defects in primary cilia formation have been implicated in a number of genetic disorders, such as Bardet-Biedl Syndrome and Polycystic Kidney Disease. Recent studies have demonstrated that polarized vesicular transport involving Rab8 and its guanine nucleotide-exchange factor Rabin8 is essential for primary ciliogenesis. Here we report that Rabin8 is a direct downstream effector of Rab11, which functions in membrane trafficking from the trans-Golgi network and recycling endosomes. Rab11, in its GTP- bound form, interacts with Rabin8 and kinetically stimulates the guanine nucleotide-exchange activity of Rabin8 toward Rab8. Rab11 is enriched at the base of the primary cilia and inhibition of Rab11 function by a dominant-negative mutant or RNA interference blocks primary ciliogenesis. Our results suggest that Rab GTPases coordinate with each other in the regulation of vesicular trafficking during primary ciliogenesis. A highly conserved region of Rabin8 is pivotal for its activation as a guanine nucleotide exchange factor for Rab8. In addition, in its activated conformation, Rabin8 interacts with Sec15, a subunit of the exocyst and downstream effector of Rab8. Expression of constitutively activated Rab8 promotes the association of Sec15 with Rabin8. Sec15 co-localized with Rab8 along the primary cilium. Inhibition of Sec15 function in cells led to defects in primary ciliogenesis. The Rabin8-Rab8-Sec15 interaction may couple the activation of Rab8 to the recruitment of the Rab8 effector and is involved in the regulation of vesicular trafficking for primary cilium formation.
Projektbezogene Publikationen (Auswahl)
- Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc Natl Acad Sci USA. 2010 Apr 6;107(14):6346-51
Knödler A, Feng S, Zhang J, Zhang X, Das A, Peränen J, Guo W.
- Structure-function study of the N-terminal domain of exocyst subunit Sec3. J Biol Chem. 2010 Apr 2;285(14):10424-33
Baek K, Knödler A, Lee SH, Zhang X, Orlando K, Zhang J, Foskett TJ, Guo W, Dominguez R
- A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis. J Biol Chem. 2012 May 4;287(19):15602-9
Feng S, Knödler A, Ren J, Zhang J, Zhang X, Hong Y, Huang S, Peränen J, Guo W
