THE LISTERIA MONOCYTOGENES-SECRETED P60 PROTEIN IS AN N-END RULE SUBSTRATE IN THE CYTOSOL OF INFECTED-CELLS - IMPLICATIONS FOR MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I ANTIGEN-PROCESSING OF BACTERIAL PROTEINS

Cytosolic antigen degradation is an initial step in the generation of major histocompatibility complex (MHC) class I-associated cytolytic T lymphocyte epitopes, Intracellular Listeria monocytogenes secretes p60 , a murein hydrolase, into the host cell cytosol, where it is degraded by proteasomes, Roughly 3% of degraded p60 gives rise to p60 217-225, a nonamer peptide that is bound by H-2K(d) MHC class I molecules. Her ein, we introduce targeted deletions throughout the p60 gene to identi fy potential proteolytic signals within p60. Degradation of mutant for ms of p60 was investigated in macrophages infected with recombinant L. monocytogenes, We found that deletions within the amino-terminal two- thirds of p60 enhanced cytosolic degradation, In contrast, truncation of the C terminus resulted in modest stabilization of p60 in the host cell cytosol, Because a protein's N-terminal amino acid can determine its rate of degradation, we mutagenized this residue in p60 into known stabilizing and destabilizing residues, Valine substitution dramatica lly stabilized cytosolic p60 molecules, while substitution with aspart ic acid resulted in rapid degradation, The number of p60 217-225 epito pes isolated from infected cells directly correlated with the rates of p60 degradation, Our data, therefore, indicate that the N-terminal am ino acid and multiple internal regions of p60 influence its stability in the cytosol of infected cells, Antigen degradation and epitope gene ration are linked, and different degradation signals can channel bacte rial proteins into the MHC class I antigen processing pathway.