The proteasome controls the degradation of the α3, β2, and β4 nAChR subunits in PC12 cells. Differentiated PC12 cells were treated with the proteasome inhibitor, PS-341, or the lysosome inhibitor, E-64, in the presence of emetine, a protein synthesis inhibitor. a, b Probing of cell lysates with anti-ubiquitin yielded a classical ladder of ubiquitinated proteins (a) which indicated that PS-341 can significantly (b) increase the total levels of ubiquitinated proteins in PC12 cell lysates. c. PS-341 inhibited proteasomal chymotrypsin-like activity by ~73%. E-64 also induced a ~11% reduction in chymotrypsin-like activity. d–g Exposure of the cells to PS-341 (d) resulted in a 2-fold increase in total levels of α3 (e), β2 (f), and β4 (g) over the three sets of experiments we conducted to examine each nAChR subunit. There was a trend toward increased levels of subunits also in the presence of E-64, but the results did not achieve statistical significance. *p≤0.05; **p≤0.005; ***p≤0.001

The proteasome inhibitor PS-341 increases the levels of the α3, β2, and β4 nAChR subunits in GM-130 positive cell fractions. Cell lysates were prepared from three batches of differentiated PC12 cells treated with vehicle or PS-341 for 24 h in the presence of emetine. Cell lysates were fractionated by centrifugation, using an iodixanol step gradient (8–34% linear Iodixanol gradient) to separate the ER/Golgi and plasma membrane compartments. Twenty fractions were collected from each cell lysate, and equal volumes of proteins from each fraction were analyzed by SDS-PAGE and immunoblotting. The blots were probed with antibodies against either α3 (a), β2 (b), or the β4 (c) nAChR subunit and the GM-130 Golgi marker (bottom blots in a–c). Cell treated with the proteasome inhibitor displayed nAChR upregulation in the fractions enriched with GM-130. The level of the α3 and β2, but not the β4, nAChR subunits was also increased in plasma membrane-enriched fractions (marked with asterisk). d Fractions enriched for the Golgi compartment (fractions 15–17) were subjected to p62-pull down followed by SDS-PAGE and probing with anti-α3 antibody. The data show an accumulation of ubiquitinated α3 in the presence of PS-341

The α3, β2, and β4 nAChR subunits are ubiquitinated. Differentiated PC12 cells, which endogenously express α3β2 and α3β4 nAChRs, were treated with emetine for 24 h in the presence or absence of PS-341. Cells lysates were incubated with anti-α3, anti-β2, or anti-β4 nAChR antibodies immobilized on protein A/G agarose beads. After SDS-PAGE separation and blotting with anti-ubiquitin antibodies, we detected a ladder of ubiquitinated subunits. Ubiquitinated species appear as clusters of high molecular weight bands (marked with bracket)

Protein degradation pathways and nAChR trafficking. Inhibition of the 26S proteasome can facilitate the trafficking of fully assembled nAChRs toward the plasma membrane. ERAD I is the main degradation pathway at the ER level while the lysosome might participate in nAChR regulation at the Golgi level and, to a lesser extent, at the ER level (ERAD II)