Abstract
SummaryAntigen presentation and microbial killing are critical arms of host defense that depend upon cargo trafficking into lysosomes. Yet, the molecular regulators of traffic into lysosomes are only partly understood. Here, using a lysosome-dependent immunological screen of a trafficking shRNA library, we identified the Arf-like GTPase Arl8b as a critical regulator of cargo delivery to lysosomes. Homotypic fusion and vacuole protein sorting (HOPS) complex members were identified as effectors of Arl8b and were dependent on Arl8b for recruitment to lysosomes, suggesting that Arl8b-HOPS plays a general role in directing traffic to lysosomes. Moreover, the formation of CD1 antigen-presenting complexes in lysosomes, their delivery to the plasma membrane, and phagosome-lysosome fusion were all markedly impaired in Arl8b silenced cells resulting in corresponding defects in T cell activation and microbial killing. Together, these results define Arl8b as a key regulator of lysosomal cellular and immunological functions.
Highlights
Intracellular trafficking to and from lysosomes is a key event in many processes required for host defense
The tails of CD1b and murine CD1d (mCD1d) bind adaptor protein 3 (AP-3), which sorts them into late endosomes and lysosomes (Cernadas et al, 2003; Chiu et al, 2002; Elewaut et al, 2003)
Given that library screening was performed with the U937 tumor cell line, we extended this finding by using primary human monocyte-derived dendritic cells (DCs) as professional antigen-presenting cells (APCs)
Summary
Intracellular trafficking to and from lysosomes is a key event in many processes required for host defense. After synthesis in the endoplasmic reticulum (ER) and delivery to the cell surface, CD1 molecules are internalized into the endocytic system where they bind lipid antigens and carry them back to the cell surface to stimulate T cell activation (Cohen et al, 2009). Previous studies on CD1 trafficking defined tyrosine-based sorting motifs in the tails of CD1 isoforms (CD1b, CD1c, and CD1d), which bind adaptor protein 2 (AP-2) and mediate their internalization into the early endocytic system via clathrin-coated pits. The tails of CD1b and mCD1d bind adaptor protein 3 (AP-3), which sorts them into late endosomes and lysosomes (Cernadas et al, 2003; Chiu et al, 2002; Elewaut et al, 2003). Entry into lysosomes is critical for access to saposins, which load lipids into CD1 molecules, and for access to degradative enzymes that processes microbial lipid antigens (Cohen et al, 2009)
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