Tuning the Cellular Trafficking of the Lysosomal Peptide Transporter TAPL by its N-terminal Domain

Abstract

The homodimeric ATP-binding cassette (ABC) transport complex TAPL (transporter associated with antigen processing-like, ABCB9) translocates a broad spectrum of peptides from the cytosol into the lumen of lysosomes. The presence of an extra N-terminal transmembrane domain (TMD0) lacking any sequence homology to known proteins distinguishes TAPL from most other ABC transporters of its subfamily. By dissecting TAPL, we could assign distinct functions to the core complex and TMD0. The core-TAPL complex, composed of six predicted transmembrane helices and a nucleotidebinding domain, is sufficient for peptide transport, showing that the core transport complex is correctly targeted to and assembled in the membrane. Strikingly, in contrast to the full-length transporter, the core translocation complex is targeted preferentially to the plasma membrane. However, TMD0 alone, comprising a putative four transmembrane helix bundle, traffics to lysosomes. Upon coexpression, TMD0 forms a stable non-covalently linked complex with the core translocation machinery and guides core-TAPL into lysosomal compartments. Therefore, TMD0 represents a unique domain, which folds independently and encodes the information for lysosomal targeting. These outcomes are discussed in respect of trafficking, folding and function of TAPL.

The lysosomal polypeptide transporter TAPL is stabilized by the interaction with LAMP-1 and LAMP-2

“…MHC class II molecules were also detected by tandem affinity purification of core-TAPL implying that the core complex is sufficient for this interaction, which is not restricted to the lysosomal compartment. Strikingly, TAPL and intracellular MHC class II expression overlaps (Demirel et al, 2010) and the expression of TAPL is strongly upregulated in professional antigen presenting cells (Demirel et al, 2007). Therefore, it can be speculated that in professional antigen presenting cells the interaction with MHC class II molecules has a functional impact on loading of antigenic peptides derived from cytosol by TAPL increasing the diversity of antigenic peptides.…”

“…2C, Fig. 3B) indicating that the unique N-terminal TMD0 of TAPL, which redirects core-TAPL by non-covalent interactions into lysosomes (Demirel et al, 2010), could represent the interaction domain. To prove this hypothesis, we stably expressed TMD0 (residues 1-142) containing a C-terminal myc-tag in wild type, LAMP-1 and/or LAMP-2 deficient mouse embryonic fibroblasts by retroviral transduction (Fig.…”

“…Cloning of human wt-TAPL, core-TAPL (aa 143-766 containing an N-terminal methionine) and TMD0 (aa 1-142 containing a C-terminal myc-tag) into pLPCX and pMSCV-IRES-Thy1.1 vectors were described previously (Demirel et al, 2010). wt-TAPL or core-TAPL was cloned into pZome-1-C (Euroscarf, University of Frankfurt a.M., Germany) resulting in constructs with a C-terminal tandem affinity purification tag consisting of calmodulin binding peptide and protein A separated by a TEV cleavage site.…”

“…Each subunit contains an N-terminal transmembrane domain and a C-terminal, cytosolic nucleotide binding domain (NBD). Functionally, TAPL can be dissected into TMD0, which shows no sequence homology to proteins in the data bases, and the core complex (Demirel et al, 2010). Core-TAPL, composed of the six putative C-terminal transmembrane helices and the NBD, is fully active in peptide transport but mistargeted predominantly to the plasma membrane when expressed alone.…”

“…Core-TAPL, composed of the six putative C-terminal transmembrane helices and the NBD, is fully active in peptide transport but mistargeted predominantly to the plasma membrane when expressed alone. TMD0, which comprises the four N-terminal putative transmembrane helices, encodes the targeting domain for lysosomal localization (Kamakura et al, 2008) and is able to redirect the core complex to lysosomes by non-covalent interactions (Demirel et al, 2010).…”