McNiven, L. as well as the role of its CT. We found that CD1a can be internalized via a clathrin- and dynamin-independent pathway and that it follows a Rab22a- and ARF6-dependent recycling pathway, similarly to other cargo internalized independently of clathrin. We also found that the CD1a CT is S-acylated. However, this Ac-IEPD-AFC post-translational modification does not determine the rate of internalization or recycling of Ac-IEPD-AFC the protein, or its localization to detergent-resistant membrane microdomains (DRM), where we found CD1a to be enriched. We also show that plasma membrane DRM are essential for efficient CD1a-mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen-loading compartments, among CD1 isoforms. Furthermore, we identify CD1a as Ac-IEPD-AFC a new marker for the clathrin- and dynamin-independent and DRM-dependent pathway of internalization as well as the Rab22a- and ARF6-dependent recycling pathway. type, in which Y is a tyrosine, any amino acid Ac-IEPD-AFC and a bulky hydrophobic residue. In contrast, CD1a does not contain any recognizable sorting motifs. The deletion of the CTs of CD1b and murine CD1d reduces or abolishes the internalization and trafficking of the respective proteins to late endocytic compartments (8-12). Moreover, tail-deleted CD1b and CD1d mutants display an impaired antigen-presenting capacity (9, 11, 13). In addition, some CD1 proteins possess putative dileucine-based sorting motifs (14), although it has not been shown if these play any role in trafficking or function. Tyrosine- and dileucine- based motifs are known to bind adaptor protein complexes (AP), such as AP-2, which is involved in endocytosis [reviewed in (15)]. In fact, Ac-IEPD-AFC human CD1b, CD1c and murine CD1d CTs have been shown to bind AP-2 or Rabbit polyclonal to FARS2 one of its subunits (8, 12). This last finding implies that CD1b, CD1c and CD1d are internalized in an AP-2-dependent manner, a pathway that is also mediated by clathrin, which coats the endocytic vesicles, and dynamin, which is thought to be involved in membrane fission. Indeed, CD1b internalization has been shown to be dynamin-dependent (8). Despite lacking any known sorting motifs in its CT, CD1a is internalized from the plasma membrane into endosomal compartments and recycles back to the plasma membrane, through the ERC (7, 16). It is not clear, however, if the CD1a CT determines the intracellular trafficking pathways followed by this molecule. Here, we investigated the internalization and recycling pathways followed by CD1a and the role of the CD1a CT. Our results suggest that CD1a and MHC class I follow similar trafficking pathways within the endocytic system. RESULTS CD1a follows a clathrin-, AP-2- and dynamin-independent pathway of internalization CD1a was previously found in clathrin-coated pits and clathrin-coated vesicles (7, 16). However, unlike other CD1 isoforms, CD1a does not possess a tyrosine-based motif or other known sorting motifs that can bind AP-2 and mediate clathrin-dependent internalization (Table 1). Therefore, we investigated the pathway of internalization followed by CD1a. We first utilized a knock-down approach directed at the clathrin-heavy chain (CHC) and the 2 2 subunit of AP-2 using siRNA. For this, HeLa cells stably expressing CD1a or CD1b were transfected with siRNA oligos and analyzed for the presence of CD1 on the surface by flow cytometry (Figure 1, A and B). As a control, transferrin receptor (TfR), a molecule known to follow a clathrin-mediated pathway of internalization was used. The TfR showed a marked increase in surface.