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Discovery of New Cargo Proteins that Enter Cells
through Clathrin-Independent Endocytosis
Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie Porat-Shliom, Roberto Weigert, Wells W. Wu, Rong-Fong Shen and Julie G.
Donaldson
Presented by:Marjolein HaagsmanNatasja Schoonen
Supervised by: Dr. P. van Bergen en Henegouwen
Traffic 2009; 10: 590-599
Arf6 regulated CIE
• Independently of clathrin & dynanim
• Requires free cholesterol
• Regulated by GTP-binding proteins (Arf6)– Alters cortical actin cytoskeleton– Alters lipid composition PM– Arf6Q67L:
• Stimulation CIE• Accumulation cargo in vacuolar-type structure
CIE vs CDE
Route depends
on specific
targeting
sequences at
cargo proteins
Research aim
• Understanding the machinery responsible for ARF6 mediated endocytosis
• By identifying PM proteins that enter the cells through the Arf6 route
How?
Isolating early endosomes originating from A6-CIE
• Use of a receptor for this route: MHC1 and intracellular marker: GFP-syn3
• Inhibit trafficking: Arf6-Q67L
• Preparation cell lysates
• Affinity purification with anti-GFP-coupled magnetic beads
Isolating early endosomes originating from A6-CIE
Identifying new proteinsby LC-MS/MS
Co-localization of identified proteins within Arf6 route?
Transfected with:
MEM-GFP: marker Arf6 route
Arf6Q67L
Transfected with:
MEM-GFP: marker Arf6 route
Arf6Q67L
Co-localization of identified proteins within Arf6 route?
MHC1 and Tfn colocalize with CD55 and Glut1
60 min.
60 min.
CD44, CD98 and CD147 colocalizes partly with MHC1 and not with Tfn
Recap
• Identification of new cargo proteins in CIE
• New cargo proteins differ in their intracellular trafficking
CD98 MHC1 colocalization varies during internalization
CD55, MHC1, Glut1 traffic via EEand CD98, CD147 and CD44 not
CD55 HA-Glut1MHC1
CD98 CD147 CD44
EEA1
EEA1
Recap
• CD98 internalizes faster than MHCI
• Different route of internalization between CD55/Glut1 and CD44/98/147
MHC1 and CD98 internalize dynamin independent
K44 = dynamin2 mutant
Filipin binds PM cholesterol
MHC1 and CD98 internalize cholesterol dependent
Conclusion
Future research
• How are CIE vesicles formed?
• Cellular function of CIE pathways?
Discussion
• Why not EM pictures?
• Why not inhibiting clathrin?
• Why cell lysates?