Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site.
In previous work, we showed that peptides from endocytosed proteins containing the tyrosine YXXphi sorting motif are recognized by the mu 2 subunit of AP-2, the plasma membrane clathrin adaptor protein complex. This interaction is activated by phosphoinositide lipids that are phosphorylated at the D-3 position of the inositol ring, and is also enhanced by the formation of clathrin-AP-2 coats. Here, we describe the detection of a specific interaction between peptides containing a second sorting motif, the dileucine motif, and AP-1, the clathrin adaptor complex responsible for sorting proteins at the trans-Golgi network (TGN). Surprisingly, the site of dileucine binding is the beta1 subunit, not mu 1. A YXXphi-containing peptide from a protein trafficked within the TGN does bind to mu 1, however. Phosphatidylinositol 3,4-diphosphate and 3,4, 5-triphosphate did not activate the interaction between dileucine-containing peptides and AP-1 but instead inhibited it, and clathrin-AP-1 coat formation did not alter the interaction. Thus, there are at least two physically separate binding sites for sorting signals on APs, which are also regulated independently.
Rapoport
I
I
Department of Cell Biology, Harvard Medical School and The Center for Blood Research.
Chen
Y C
YC
Cupers
P
P
Shoelson
S E
SE
Kirchhausen
T
T
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D017252:CD3 Complex / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism; D002417:Cattle; D002966:Clathrin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005615:Freezing; D051275:Glucose Transporter Type 4; D006023:Glycoproteins; D007930:Leucine / Q000378:metabolism*; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D010455:Peptides / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D015703:Antigens, CD; D017252:CD3 Complex; D015704:CD4 Antigens; D002966:Clathrin; D004268:DNA-Binding Proteins; D051275:Glucose Transporter Type 4; D006023:Glycoproteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010455:Peptides; D018129:Phosphatidylinositol Phosphates; D017527:Receptor, IGF Type 2; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D014157:Transcription Factors; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D007930:Leucine