Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie.

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?