AP-1 and retromer play opposite roles in the trafficking of sortilin between the Golgi apparatus and the lysosomes Maryssa Canuel a , Stephane Lefrancois b , Jibin Zeng a , Carlos R. Morales a, * a Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montreal, Que., Canada H3A 2B2 b Centre de Recherche de l’hopital Maisonneuve-Rosemont, Montreal, Que., Canada Received 23 November 2007 Available online 17 December 2007 Abstract Sortilin has been implicated in the sorting of one soluble hydrolase and two sphingolipid activator proteins to the lysosomes. While the GGA adaptor proteins have been demonstrated to play a role in the targeting of sortilin to the endosomes, the recycling of sortilin has not yet been elucidated. Here we examine the role of two adaptor protein complexes, AP-1 and retromer. Our results demonstrate that AP-1 is required for the transport of sortilin to the endosomes and retromer for the recycling of sortilin to the Golgi apparatus. While inhibition of AP-1 causes accumulation of sortilin in the Golgi apparatus, RNAi depletion of retromer results in retention of sor- tilin in the lysosomes. We also demonstrate that the interaction of sortilin with retromer occurs through a YXXU site in its cytosolic tail. In conclusion, our observations indicate that retromer and AP-1 play opposite roles in the trafficking of sortilin. Crown copyright Ó 2007 Published by Elsevier Inc. All rights reserved. Keywords: Sorting receptors; Sortilin; Retromer; AP-1; Prosaposin Efficient delivery of lysosomal sorting receptors and their cargo depends upon interaction with adaptor proteins [1–5]. Following release of their cargo into the endosomal lumen, sorting receptors are suspected to recycle back to the Golgi via the retromer complex [6,7,10]. The mamma- lian retromer complex consists of Vps26, Vps29, Vps35, and sorting nexins (SNX) 1 and 2 [8,11]. The mannose 6-phosphate receptor (M6PR), responsi- ble for sorting most soluble hydrolases [12,13], has been demonstrated to reach the endosomal compartment through interactions with the Golgi-localized, c ear-con- taining ARF binding adaptor proteins (GGAs), as well as with AP-1 [14–19]. AP-1 is a heterotetrameric adaptor that consists of two large subunits (c and b1), a medium subunit (l1), and a small subunit (r1) [20]. The b-subunit of AP-1 has been demonstrated to interact with clathrin, while interaction with the receptor occurs through the l-subunit [21–23]. Upon release of its cargo, the M6PR binds retro- mer and recycles back to the Golgi apparatus [24]. The interaction between retromer and the M6PR is purported to occur through the Vps35 subunit of retromer [24]. Sortilin is a sorting receptor that has been implicated in the trafficking of sphingolipid activator proteins (SAPs) [25]. Interestingly, the cytoplasmic tails of the M6PR and sortilin exhibit sequence and functional homology [27]. These observations have led us to hypothesize that the M6PR and sortilin share a conserved sorting and traffick- ing mechanism. Herein we demonstrate a role for both retr- omer and AP-1 in the trafficking of sortilin. We also identify a new sequence in the cytoplasmic tail of sortilin that governs the interaction with retromer. Materials and methods Cell culture. COS-7 cells were cultured in DMEM (Invitrogen) sup- plemented with 10% FBS, 5% penicillin and streptomycin, and L-gluta- mine. Cells were maintained at 5% CO 2 at 37 °C. The cells were 0006-291X/$ - see front matter Crown copyright Ó 2007 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.015 * Corresponding author. Fax: +1 514 398 5047. E-mail address: carlos.morales@mcgill.ca (C.R. Morales). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 366 (2008) 724–730