Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding peptide into the endoplasmic reticulum in an ATP-dependent manner.

Abstract

We have investigated the role of the putative peptide transporters associated with antigen processing (TAP) by using a permeabilized-cell system. The main objective was to determine whether these molecules, which bear homology to the ATP-binding cassette family of transporters, translocate antigenic peptides across the endoplasmic reticulum membrane for assembly with major histocompatibility complex (MHC) class I molecules and P2-microglobulin light chain. The poreforming toxin streptolysin 0 was used to generate permeabilized cells, and peptide translocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptide was associated with MHC class I glycoproteins from unpermeabilized cells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependent. In antigen-processing mutant cells lacking a functional transporter, uptake occurs only through a less-efficient transporter and ATP-independent pathway. In addition, short peptides (8-10 amino acids) known to bind MHC class I molecules compete efficiently with a radiolabeled peptide for TAP-dependent translocation, whereas longer peptides and a peptide derived from an endoplasmic reticulum signal sequence do not compete efficiently. This result indicates that the optimal substrates for TAP possess the characteristics of MHC-binding peptides.A basic requirement for the presentation ofantigenic peptides by major histocompatibility complex (MHC) class I molecules to T cells is the transport of these peptides to the site of class I assembly. The breakdown of protein antigens to peptides destined for presentation by class I molecules is thought to occur in the cytosol through the degradative action of proteasomes (1). Two proteins encoded within the MHC, termed LMPs for low molecular weight proteins, share homology with proteasome subunits and may play a role in the specific degradation of MHC class I-associated antigens (2). Once the peptide is generated, it must be targeted to the correct compartment where it can bind to class I molecules. The available evidence suggests that this compartment is the endoplasmic reticulum (ER) (3). The discovery of two other proteins encoded within the MHC, termed TAP-1 and TAP-2 for transporters associated with antigen processing because of their homologies to the ATP-binding cassette family of transporters (for review, see refs. 2 and 4), has led to speculation that these proteins are responsible for transporting peptides across the ER membrane. Restoration of normal class I processing and presentation occur in mutant cell lines that lack one or both of the TAP proteins when functionalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. TAP-encoding genes are introduced by transfection (5, 6).Two such mutant cell lines, .174 a...

Intracellular peptide transporters in human – compartmentalization of the “peptidome”

“…Peptide binding is ATP-independent [88,90], whereas, peptide translocation requires ATP hydrolysis [3,61,65,79]. Peptide binding is composed of a fast association step followed by a slow structural reorganization of the TAP complex [68].…”

Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment.

“…The resultant peptides are then translocated into the lumen of the RER by the transporter associated with antigen presentation (TAP) (Powis et al, 1991;Spies and DeMurs, 1991;Attaya et al, 1992). TAP is a heterodimer composed of two subunits (TAP1 and TAP2) located in the ER and cis-Golgi , and is responsible for the translocation of peptides of particular size and sequence (Androlewicz et al, 1993;Neefjes et al, 1993;Shepherd et al, 1993;Heemels and Ploegh, 1995).…”

Reconstitution of peptide‐binding activity by TAP in proteoliposomes

“…The peptides were iodinated as previously described [9]. Briefly, 20 nmoles of peptide was iodinated using the method of chloramine T, and the unbound iodine was removed by gel filtration on a Sephadex G-10 column.…”

“…Proteoliposome pellets, derived from 4.1 mg total membrane protein, were resuspended in 500 μΐ intracellular transport (ICT) buffer (50 mM HEPES, pH 7.0, 78 mM KC1, 4 mM MgCl 2 , 8.4 mM CaCl 2 , 10 mM EGTA, 1 mM DTT, and 4 mg/ml bovine serum albumin, see [9]). 125 I-B27#3 (100 nM) was added to the proteoliposome suspension and allowed to incubate for 20 min on ice.…”

“…in permeabilized cells [9,10] and microsomes [11,12]. Although these studies have produced valuable information on how TAP works, in order to understand TAP function in greater molecular detail, the functional reconstitution of TAP into artificial membranes will be necessary.…”