Two novel routes of transporter associated with antigen processing (TAP)-independent major histocompatibility complex class I antigen processing

Abstract

Jaw1 is an endoplasmic reticulum (ER) resident protein representative of a class of proteins post translationally inserted into membranes via a type II membrane anchor (cytosolic NH2 domain, lumenal COOH domain) in a translocon-independent manner. We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein. Peptide delivery mediated by Jaw1 to class I molecules was equal or better than that mediated by the adenovirus E3/19K glycoprotein signal sequence, and was sufficient to enable cytofluorographic detection of newly recruited thermostabile class I molecules at the surface of TAP-deficient cells. Deletion of the transmembrane region retargeted Jaw1 from the ER to the cytosol, and severely, although incompletely, abrogated its TAP-independent peptide carrier activity. Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1. These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.

Figure 1

(A) Schematic representation of rVV expressed Jaw1 chimeric proteins. (B) Biochemical characterization of Jaw1 chimeric proteins. Species reactive with anti-Jaw1 antisera present in detergent extracts of rVV-infected cells pulse radiolabeled and chased for the indicated time in hours were analyzed by SDS-PAGE and visualized using a PhosphorImager. Only the region containing the antibody-reactive species is shown. The far left lane contains molecular weight markers indicated. The asterisk identifies a proteolytic fragment created by the action of Kex2 on Jaw1[NP_147–155].

Figure 1

(A) Schematic representation of rVV expressed Jaw1 chimeric proteins. (B) Biochemical characterization of Jaw1 chimeric proteins. Species reactive with anti-Jaw1 antisera present in detergent extracts of rVV-infected cells pulse radiolabeled and chased for the indicated time in hours were analyzed by SDS-PAGE and visualized using a PhosphorImager. Only the region containing the antibody-reactive species is shown. The far left lane contains molecular weight markers indicated. The asterisk identifies a proteolytic fragment created by the action of Kex2 on Jaw1[NP_147–155].

Figure 6

Effect of protease inhibitors on generation of the NP_147–155 peptide. rVV-infected T2-K^d cells expressing the indicated protein were incubated with the indicated protease inhibitor starting 30 min before infection and ending 4 h after infection. BFA was then added to cells to prevent additional antigen presentation and cells were incubated with NP_147–155-specific T_CD8+ in a standard microcytotoxicity assay at the effector to target ratios indicated. Inhibitors were used at the following concentrations: NH₄Cl, 25 mM; cbz–LL–CH, 12.5 μM; cbz–LLL–CHO 12.5 μM (A and B), 25 μM (C); lactacystin. 10 μM; N–Ac–LLnL, 200 μM; N–Ac–LLnM, 200 μM. The three panels are derived from separate experiments.

Figure 2

Immunogold localization of Jaw1 proteins. Jaw1 was located in rVV-infected T2 cells by cryoimmunogold labeling. (A) Cells expressing Jaw1 (Lum⁻)[NP_147–155]. Gold particles specifically decorate the nuclear membrane and cytoplasmic membrane structures. (B) Cells expressing Jaw1 (TM⁻)[NP_147–155]. Gold particles are located in the cytosol. In addition to the difference in patterns between A and B, the specificity of Jaw staining was shown by the absence of gold particles on sections prepared from cells infected with a control rVV.

Figure 3

Presentation of NP_147–155 by rVV-infected cells. rVV-infected cells expressing the Jaw chimeric proteins were tested for lysis by NP-specific secondary T_CD8+ at the indicated effector to target ratio. Cells were coinfected with a rVV expressing K^d to enable recognition.

Figure 4

Rescue of K^d cell surface expression. T2-K^d cells were infected with the rVV indicated and the amount of cell surface K^d present on viable cells cytofluorographically determined using a directly conjugated K^d-specific mAb. Data are expressed as mean channel fluorescence × 100. The last three bars on the right represent cells coinfected with Jaw1[NP_147–155] and the rVV indicated (ACE, angiotensin-converting enzyme).

Figure 5

Quantitation of acid-soluble peptides in cell extracts. Extracts prepared from cells expressing ER retained K^d (Kd Ret) and the indicated Jaw1 chimeric protein were tested for their ability to sensitize P815 cells for lysis by NP-specific T_CD8+. Cell mix refers to a sample in which ER-retained K^d expressing cells were mixed with Jaw1(Lum⁻)[NP_147–155]- expressing cells before lysis.

Figure 7

Tap-independent presentation in nonlymphoid cells. HeLa cells coinfected with rVVs expressing K^d, a Jaw1 chimerical protein, and either ICP47 or no foreign protein were tested for recognition by T_CD8+ populations specific for NP or VV at the indicated effector to target ratio.