Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events

Abstract

Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, mu and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of mu and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all mu chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of mu chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates.

Endoplasmic Reticulum (ER)-associated Degradation of T Cell Receptor Subunits

“…How- ever, even after 16 h of proteasome inhibition, the amount of cytoplasmically disposed TCR-␣ represents only a small fraction of the total accumulated material, suggesting a continued requirement for proteasome function for efficient complete retrotranslocation and degradation from the ER, as has been suggested for an engineered model substrate (31) and for Pdr5 (8) in yeast. Similarly, a requirement for proteasome function in retrotranslocation has been reported for unassembled soluble Ig subunits (32) and CD4 (33,34) in mammalian cells. Results with TCR-␣ demonstrate that the relative amounts of cytoplasmically disposed material is consistently decreased when inactive MmUBC7 is coexpressed, even when proteasome function is inhibited, suggesting that MmUBC7 may play a role in the process leading to retrotranslocation upstream of the involvement of proteasomes.…”

Spontaneous transgenesis of human B lymphocytes

“…Plasmid g1NANP carries an insertion in the third complementarity-determining region (CDR3) of the variable domain coding for three repeats of the tetrapeptide NANP. 22 Plasmid g1MART (used in experiments shown in Figure 6) codes for the amino-acid sequence AAGIGILTV (position [27][28][29][30][31][32][33][34][35] from the MART-1 melanoma-associated antigen 23 in lieu of the three NANP repeats. Both plasmids are under the control of a B-cell-specific promoter.…”

“…28 Then a sizable fraction of newly synthesized polypeptide enters the catabolic pathway, which could be either local 29 or proteosome mediated, 30 yielding peptides that can load the groove of MHC class I molecules in the endoplasmic reticulum. 31 The peptide-MHC class I complex is then exported to the cell surface serving as target recognition for specific CD8 þ T lymphocytes.…”

“…No lysis was observed at any of the E:T ratios used (Figure 8). Therefore, lysis of transgenic cells requires expression, processing, and presentation of the MART-1 [27][28][29][30][31][32][33][34][35] peptide.…”

“…After washings, cells were resuspended in Cr-release assay EGFP þ BJAB cells expressing g1MART were generated by spontaneous transgenesis followed by culture under continuous G418 selection. After subcloning, positive cultures were identified by FACS analysis and used as a target in a conventional 51 Cr-release assay where the HLA-A2-restricted MART-1 [27][28][29][30][31][32][33][34][35] specific CTL, TIL #1235, and TIL #1088 23 (the kind gift of Dr N Restifo, National Cancer Institute, Bethesda, MD, USA) served as the effectors per our standard assay conditions. 74 The percentage of lysis was calculated as follows: 100 Â (sample cpmÀspontaneous release cpm)/(maximum releaseÀspontaneous release).…”