Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the ef®ciency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1±7 additional N-terminal residues. Only 6±8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-g-induced b-subunits and are more ef®cient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2±4 times more of certain N-extended versions. These observations show that the changes in cleavage speci®city of immunoproteasomes in¯uence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-ginduced enzyme leucine aminopeptidase).

The finding that MHC class I molecules are physically associated with the TAP transporter has suggested that peptides may be directly transported into the binding groove of the class I molecules rather than into the lumen of the endoplasmic reticulum (ER) where they subsequently would encounter class I molecules by diffusion. Such a mechanism would protect peptides from peptidases in the ER and/or escaping back into the cytoplasm. However, we find that an anti-peptide Ab that is cotranslationally transported into the ER prevents TAP-transported peptides from being presented on class I molecules. The Ab only blocks the binding of its cognate peptide (SIINFEKL) but not other peptides (KVVRFKDL, ASNENMETM, and FAPGNYPAL). Therefore, most TAP-transported peptides must diffuse through the lumen of the ER before binding stably to MHC class I molecules.

Recent studies have suggested that the membrane proximal region of the cytoplasmic domain of the erythropoietin receptor and other members of the cytokine receptor superfamily may be required for signal transduction. Expression of several deletion mutants of the erythropoietin receptor in Ba/F3 cells showed that a region with homology to the interleukin-2 receptor beta-chain which includes Box 2 is not essential for erythropoietin-dependent cell proliferation. However, a region between Box 1 and Box 2 contains essential residues for proliferative response. Expression of mutant receptors was confirmed by reverse transcriptase-PCR analysis and by Western blotting, which also showed no evidence for expression of endogenous wild-type receptor. These findings are in direct conflict with previously reported mutagenesis studies of the erythropoietin receptor suggesting that mitogenesis may be channelled through more than one pathway depending on the complement of signaling molecules expressed in the cell.