The Listeria monocytogenes-secreted p60 Protein Is an N-end Rule Substrate in the Cytosol of Infected Cells

Abstract

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. Herein, we introduce targeted deletions throughout the p60 gene to identify potential proteolytic signals within p60. Degradation of mutant forms 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 dramatically stabilized cytosolic p60 molecules, while substitution with aspartic acid resulted in rapid degradation. The number of p60 217-225 epitopes isolated from infected cells directly correlated with the rates of p60 degradation. Our data, therefore, indicate that the N-terminal amino acid and multiple internal regions of p60 influence its stability in the cytosol of infected cells. Antigen degradation and epitope generation are linked, and different degradation signals can channel bacterial proteins into the MHC class I antigen processing pathway.MHC 1 class I molecules bind peptides that are generated in the cytosol by proteasome-mediated degradation of endogenous and foreign proteins (1-3). Proteasomes are multienzyme complexes consisting of a 20 S catalytic core that associates with regulatory subunits to make a 26 S particle. Membrane-permeable proteasome inhibitors prevent the generation of most antigenic peptides and, because of a shortage of peptide, impair MHC class I egress from the endoplasmic reticulum (4 -6). The sites of proteasome cleavage can influence the efficiency of antigen processing into MHC class I-associated peptides (7-9). Two MHC-encoded proteasome subunit proteins, LMP-2 and LMP-7, and the proteasome regulator PA28 modify proteasome specificity and enhance MHC class I antigen processing (10 -14). Peptides generated in the cytosol by proteasomes are translocated into the endoplasmic reticulum by the transporter associated with antigen processing (15) and, if they conform to the appropriate motif, are bound by MHC class I molecules (16). The assembled complexes travel to the cell surface, where they are presented to CD8ϩ cytolytic T lymphocytes (CTL).Cytosolic protein degradation is generally very specific and regulated. Thus, most endogenous proteins contain or acquire degradation signals prior to proteasome-mediated destruction. One of the best characterized mechanisms for marking proteins for cytosolic degradation involves the ubiquitination of target protei...

RGS4 Is Arginylated and Degraded by the N-end Rule Pathway in Vitro

“…The reactions were incubated at 30°C, unless stated otherwise; they were terminated by the addition of SDS-sample buffer, heated at 95°C for 5 min, and fractionated by SDS-PAGE, followed by autoradiography or fluorography. 35 S in protein bands was determined using PhosphorImager (Molecular Dynamics, Sunnyvale, CA).…”

“…Ⅺ, the analogous ratio curve but for Arg-nsP4* in the presence of 1 mM Arg-␤-Ala (calculated from the data in Fig. 1B; the 4-h ratio could not be determined reliably because of too low 35 S in the band of Arg-nsP4* in the absence of dipeptides). Note the monotonous increase of the dipeptides/controls ratio for engineered N-end rule substrate but not for RGS4.…”

“…1, A and B, and 2B). The resulting 35 S-labeled proteins were visualized by SDS-PAGE and autoradiography. We searched for protein bands that were selectively enhanced in the presence, but not in the absence, of the dipeptide inhibitors.…”

“…The positions of RGS4 and Arg-nsP4* bands and of the ladder of multi-Ub-containing species are indicated on the left. B, relative amounts of 35 S-RGS4 in A were determined using PhosphorImager and were plotted as a function of incubation time. The amount of RGS4 in the reaction without MG115 at 30 min was assigned the value of 100.…”

“…Physiological N-end rule substrates were also identified among the proteins secreted into the cytosol of the mammalian cell by intracellular parasites such as the bacterium Listeria monocytogenes (35). Selective perturbation of the N-end rule pathway was reported to interfere with mammalian cell differentiation (36,37) and with limb regeneration in amphibians (38).…”