Sorting of major cargo glycoproteins into clathrin-coated vesicles.
The AP-1 and AP-2 complexes are the most abundant adaptors in clathrin-coated vesicles (CCVs), but clathrin-mediated trafficking can still occur in the absence of any detectable AP-1 or AP-2. To find out whether adaptor abundance reflects cargo abundance, we used lectin pulldowns to identify the major membrane glycoproteins in CCVs from human placenta and rat liver. Both preparations contained three prominent high molecular-weight proteins: the cation-independent mannose 6-phosphate receptor (CIMPR), carboxypeptidase D (CPD) and low-density lipoprotein receptor-related protein 1 (LRP1). To investigate how these proteins are sorted, we constructed and stably transfected CD8 chimeras into HeLa cells. CD8-CIMPR localized mainly to early/tubular endosomes, CD8-CPD to the trans Golgi network and CD8-LRP1 to late/multivesicular endosomes. All three constructs redistributed to the plasma membrane when clathrin was depleted by siRNA. CD8-CIMPR was also strongly affected by AP-2 depletion. CD8-CPD was moderately affected by AP-2 depletion but strongly affected by depleting AP-1 and AP-2 together. CD8-LRP1 was only slightly affected by AP-2 depletion; however, mutating an NPXY motif in the LRP1 tail caused it to become AP-2 dependent. These results indicate that all three proteins have AP-dependent sorting signals, which may help to explain the relative abundance of AP complexes in CCVs. However, the relatively low abundance of cargo proteins in CCV preparations suggests either that some of the APs may be empty or that the preparations may be dominated by empty coats.
Harasaki
Kouki
K
University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.
Lubben
Nienke B
NB
Harbour
Michael
M
Taylor
Marcus J
MJ
Robinson
Margaret S
MS
D000595:Amino Acid Sequence; D000818:Animals; D016827:CD8 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm; D004705:Endocytosis; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D016827:CD8 Antigens; D006023:Glycoproteins; D021382:Protein Sorting Signals; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins