ARFGAPs in COP I traffic and proteomic analysis of the Golgi complex

Background:

COP I vesicle coat proteins provide essential, conserved functions in protein traffic in the secretory pathway. Activity of the small GTPase ARF, a “master switch” protein for this pathway, is critically regulated by three GTPase-activating proteins, named ARFGAP 1-3. In human cells ARFGAPs 1-3, together provide essential and overlapping functions in COP I-dependent traffic and protein sorting. These proteins mediate Golgi-to-ER and intra-Golgi retrieval pathways, and are required for maintenance of the interphase Golgi complex. Disturbances in the COP I pathway are relevant to several human pathologies.

Project:

The project will dissect individual and global cellular functions of ARFGAPs in membrane traffic. Dominant-negative ARFGAP mutants that when expressed in cells efficiently block trafficking of a COP I model cargo, fluorescently labeled cholera toxin, have successfully been generated in the Duden lab. Furthermore, RNAi oligonucleotides that allow efficient knock-down of ARFGAP1-3, either singly or in combination, have been validated. Use of these tools will enable in-depth analysis of the COP I membrane traffic pathway. A spinning disk confocal microscopy setup and a comprehensive multi-color library of organelle marker proteins labeled with fluorescent proteins, will allow dynamic changes of organelles of the secretory pathway to be recorded and qualitatively assessed. Proteomic analyses of isolated Golgi fractions will be applied to quantitatively identify changes in proteins associated with the Golgi complex in response to experimental treatments described above. Data thus obtained combined with existing databases can be used to mine relationships at the systems level.

People in this Project

Robert Schönherr, MScDoctoral Candidate
Rainer Duden, Prof. Dr. rer. nat.Investigator
Andreas Ziegler, Prof. Dr. rer. nat.Investigator

Publications

No publications so far.

    2013

    Willett R, Kudlyk T, Pokrovskaya I, Schönherr R, Ungar D, Duden R, Lupashin V. COG complexes form spatial landmarks for distinct SNARE complexes. Nat Commun. 2013 March 5; 4: 1553

     

    2014

    Isken O, Langerwisch U, Schönherr R, Lamp B, Schröder K, Duden R, Rümenapf TH, Tautz N. Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging  J. Virol. January 2014 vol. 88 no. 1 82-98