Discovery of new cargo proteins that enter cells through clathrin-independent endocytosis.
Clathrin-independent endocytosis (CIE) allows internalization of plasma membrane proteins lacking clathrin-targeting sequences, such as the major histocompatibility complex class I protein (MHCI), into cells. After internalization, vesicles containing MHCI fuse with transferrin-containing endosomes generated from clathrin-dependent endocytosis. In HeLa cells, MHCI is subsequently routed to late endosomes or recycled back out to the plasma membrane (PM) in distinctive tubular carriers. Arf6 is associated with endosomal membranes carrying CIE cargo and expression of an active form of Arf6 leads to the generation of vacuolar structures that trap CIE cargo immediately after endocytosis, blocking the convergence with transferrin-containing endosomes. We isolated these trapped vacuolar structures and analyzed their protein composition by mass spectrometry. Here we identify and validate six new endogenous cargo proteins (CD44, CD55, CD98, CD147, Glut1, and ICAM1) that use CIE to enter cells. CD55 and Glut1 appear to closely parallel the trafficking of MHCI, merging with transferrin endosomes before entering the recycling tubules. In contrast, CD44, CD98, and CD147 appear to directly enter the recycling tubules and by-pass the merge with EEA1-positive, transferrin-containing endosomes. This divergent itinerary suggests that sorting may occur along this CIE pathway. Furthermore, the identification of new cargo proteins will assist others studying CIE in different cell types and tissues.
Eyster
Craig A
CA
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Higginson
Jason D
JD
Huebner
Robert
R
Porat-Shliom
Natalie
N
Weigert
Roberto
R
Wu
Wells W
WW
Shen
Rong-Fong
RF
Donaldson
Julie G
JG
D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002477:Cells / Q000737:chemistry / Q000378:metabolism*; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000235:genetics; D011992:Endosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002966:Clathrin; D008565:Membrane Proteins; D011506:Proteins; D014168:Transferrin