Transporter associated with antigen processing (TAP)-like (TAPL; ABCB9) is a half type ATP binding cassette (ABC) transporter with ten potential transmembrane helices followed by a conserved ABC region 1,2) that typifies the ABC transporter family.3) TAPL is named TAP-like, because of its high similarity to TAP1 (ABCB2) and TAP2 (ABCB3), both of which are subunits of TAP.1) The gene structures of TAPL, TAP1 and TAP2 are very similar,4) suggesting that all these paralogs have evolved from a common ancestral gene.
2)As anticipated from its similarity to TAP subunits, it has been demonstrated that TAPL can transport peptides.
5)In contrast to TAP, which transports substrates comprising 8-12 amino acids for antigen presentation on major histocompatibility complex (MHC) class I molecules, 6) TAPL recognizes a wide variety of peptides comprising 6 to 59 residues.5) It is likely that a homodimer of TAPL could be a functional unit, 5,7,8) whereas both TAP1 and TAP2 are obligatory subunits of TAP.
9)As for its intracellular distribution, TAP is localized to the endoplasmic reticulum (ER) and cis-Golgi membranes.
10)However, the distribution of TAPL is contradictory: transiently expressed TAPL is found on the ER 2,11) as well as lysosomal membranes, 12) whereas stably expressed TAPL is sorted to lysosomes. 13,14) Such discrepancy could be partly explained as follows; the highly expressed TAPL by means of transient transfection may saturate the transport system to lysosome in ER. Furthermore, it is also reported that the mitochondrial half type ABC transporter exports peptides with 6 to 20 residues from the matrix across the inner membrane.15) However, possible localization of TAPL on the mitochondrial membrane has not been excluded definitely. Thus determination of the intracellular localization of TAPL is important for elucidation of its physiological roles that are not known except that it is a peptide transporter.
5)In this study, we examined the intracellular distribution of TAPL tagged with a green fluorescent protein (GFP). The resulting fusion protein was stably expressed in Chinese hamster ovary (CHO)-K1 cells, and its distribution in lysosomal membranes was verified by means of fluorescence microscopy and cellular fractionation. It was further suggested that TAPL is localized to the microdomains of lysosomal membranes enriched in cholesterol. Taken together, it is clear that TAPL could function differently and separately from TAP and mitochondrial ABC transporter.
MATERIALS AND METHODS
Stable Expression of TAPL Fused with GFPThe cDNA of human TAPL12A 4,11) was amplified by means of the polymerase chain reaction (PCR) method 2) with Ampli, Taq polymerase (Roche, Switzerland) using primers TM070 (5Ј-ccagatctaaccagcagg ATGcgg-3Ј) and TM071 (5Ј-cgtc gacgcCGCcttgtgactgcc-3Ј) carrying BglII and SalI sites (un derlined) upstream of the initiation codon and downstream of the Ala-766 codon (capital letters), respectively. The amplified fragment was inserted into the pGEM ® -T Easy vector (Promega, U.S.A.). The BglII-SalI...
