Proteasomes and antigen presentation: evidence that a KEKE motif does not promote presentation of the class I epitope SIINFEKL

Abstract

Previously we proposed that stretches of alternating Lys(K) and Glu(E) in polypeptides promote the expression of nearby sequences on Class I molecules [Realini, C., Rogers, S.W., Rechsteiner, M., 1994. KEKE motifs. Proposed roles in protein–protein association and presentation of peptides by MHC class I receptors. FEBS Lett. 348, 109–113]. As a test of the KEKE hypothesis we have employed osmotic lysis of pinosomes and transfection to introduce or express various ubiquitin peptide fusion proteins in the cytosol of wild type and PA28αβγ − mouse embryo fibroblasts. KEKE or non-KEKE motifs were placed between ubiquitin and the OVA epitope SIINFEKL that was at or near the C-termini of the various fusion proteins. Measurements of surface K b–SIINFEKL complexes using the monoclonal antibody 25.-D1.16 allowed us to assess the effects of upstream KEKE motifs and PA28 status on SIINFEKL surface presentation. KEKE motifs did not enhance presentation of the OVA epitope. However, our studies did confirm that PA28αβ is needed for efficient SIINFEKL surface expression when hsp90 is inhibited.