Endocytosis of synaptotagmin 1 is mediated by a novel, tryptophan-containing motif.
The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2-binding site previously described. Internalization through the tryptophan-based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan-to-phenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.
Jarousse
Nadine
N
University of California San Francisco, Genentech Hall, 600 16th St., San Francisco, California 94143-2140, USA. jarousse@cgl.ucsf.edu
Wilson
Joshua D
JD
Arac
Demet
D
Rizo
Josep
J
Kelly
Regis B
RB
D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D034281:Dynamins / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D050863:Synaptotagmin I; D050857:Synaptotagmins; D014364:Tryptophan / Q000378:metabolism*
D033962:Adaptor Protein Complex 2; D048868:Adaptor Proteins, Signal Transducing; D015704:CD4 Antigens; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C494310:SYT1 protein, human; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D014364:Tryptophan; D034281:Dynamins