Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.
Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Anand
A
Sitaram
Megan K
MK
Dennis
Rittik
R
Chaudhuri
Wilfredo
W
De Jesus-Rojas
Danièle
D
Tenza
Subba Rao Gangi
SR
Setty
Christopher S
CS
Wood
Elena V
EV
Sviderskaya
Dorothy C
DC
Bennett
Graça
G
Raposo
Juan S
JS
Bonifacino
Michael S
MS
Marks
D033961:Adaptor Protein Complex 1 / Q000378:metabolism*; D033963:Adaptor Protein Complex 3 / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004151:Dipeptides / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D037102:Lectins / Q000378:metabolism*; D008544:Melanocytes / Q000378:metabolism; D020460:Melanosomes / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D021382:Protein Sorting Signals; D021381:Protein Transport
D033961:Adaptor Protein Complex 1; D033963:Adaptor Protein Complex 3; C513476:Bloc1s6 protein, mouse; D002352:Carrier Proteins; D004151:Dipeptides; D047908:Intracellular Signaling Peptides and Proteins; D037102:Lectins; D026901:Membrane Transport Proteins; C489600:OCA2 protein, human; D021382:Protein Sorting Signals; C038361:leucylleucine