Transporter Associated with Antigen Processing-Like (ABCB9) Stably Expressed in Chinese Hamster Ovary-K1 Cells Is Sorted to the Microdomains of Lysosomal Membranes

Abstract

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...

Intracellular Localization of ABC Transporter TAPL Differs between Transient and Stable Expression

“…TAPL( )-GFP as well as TAPL-DsRed expressed stably in CHO-K1 cells were co-localized with a lysosome marker protein, cathepsin D, rather than an endoplasmic reticulum marker, PDI. This localization is consistent with our previous findings [4] [10], and that for a SKOV3 cell line derived from human ovaries [18]. The expression level would be too high upon transient expression, resulting in inhibition of the normal targeting pathway from the ER to lysosomes because various components for lysosomal targeting [19], limiting amounts of which are present in the ER, could be saturated by the excess TAPL molecules.…”

“…The downstream six transmembrane helices and the ABC domain of TAPL form a core domain essential for homo-dimerization, while its N-terminal four transmembrane helices do not participate in the dimer formation by TAPL [4]. When the fusion protein comprising TAPL with the green fluorescent protein (GFP) at its C-terminus was stably expressed in Chinese hamster ovary (CHO)-K1 cells, it was localized on the lysosomal membrane [4] [10]. It is further suggested that the N-terminal four transmembrane helices of TAPL comprise an unidentified lysosomal targeting signal [4].…”

Profiling of differentially expressed genes in sheep T lymphocytes response to an artificial primary Haemonchus contortus infection

“…Though the function of the transporter has yet to be determined, ABCB1, another member of the MDR/TAP subfamily, shows correlation with migration of immune cells and adaptive immunity [ 38 , 39 ]. Fujimoto speculated that more ABCB9 might help to regulate antigen presentation and multidrug resistance [ 40 ]. Increased expression of MCP1 encoded mast cell protease 1 would contribute to the clearance of gastrointestinal nematode [ 41 ].…”

Whole genome sequencing identified genomic diversity and candidated genes associated with economic traits in Northeasern Merino in China

“…Immunity is also an important component and aspect of environmental adaptability and several candidate genes related to those were detected. The membrane-associated protein encoded by the ABCB9 gene is associated with antigen processing ( Fujimoto et al, 2011 ). GALNT8 is related to innate and acquired immune responses and cytokine signalling, which are important for protecting sheep from parasitic invasion ( Al Kalaldeh et al, 2019 ).…”