Di-leucine signals mediate targeting of tyrosinase and synaptotagmin to synaptic-like microvesicles within PC12 cells.
One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.
Blagoveshchenskaya
A D
AD
Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom.
Hewitt
E W
EW
Cutler
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D006735:Horseradish Peroxidase; D007930:Leucine / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015398:Signal Transduction; D013572:Synaptic Vesicles / Q000378:metabolism*; D016708:Synaptophysin / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins
D015704:CD4 Antigens; D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D016708:Synaptophysin; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D006735:Horseradish Peroxidase; D014442:Monophenol Monooxygenase; D007930:Leucine