Consequences of CsA-treatment on disposal of ERAD-LM and ERAD-LS substrates.
A Labeled BACE457, an ERAD-LM substrate, was immunoisolated from total cell extracts with a specific antibody after 10 (lane 1, initial amount) or 120 min of chase (lane 2, residual amount). Labeled BACE457 was also immunoisolated from cells exposed for 120 min to CsA (lane 3), Tg (lane 4), Kif (lane 5) or PS341 (lane 6). B Quantification of the labeled polypeptide bands shown in the gel. Reproducibility of these data (i.e. lack of CsA inhibition) is confirmed by the independent experiment shown in Figs. 2B–2C, lanes 1–3. C Same as A for BACE457Δ, an ERAD-LS substrate. Chase times are 10 (initial) and 75 min (residual). The apparent mass of BACE457Δ is reduced by the progressive and extensive de-mannosylation of the protein-bound oligosaccharides during the chase (lane 1 vs 2 , , ). Consistently, enhancement in electrophoretic mobility is specifically inhibited by Kif (lane 5 in Figs. 1A and 1C; lane 4 in Figs. 1E and 1G ). CsA, Tg and PS341 inhibit BACE457Δ disposal without affecting the enhancement of electrophoretic mobility during the chase. Thus, they all affect events occurring after substrate de-mannosylation. D Same as B for BACE457Δ. The reproducibility is confirmed in Figs. 2D–2E, lanes 1–3. E Same as A for CD3δΔ, an ERAD-LS substrate lacking cis peptidyl-prolyl bonds. F Same as B for CD3δΔ. The reproducibility is confirmed in Figs. 2F–2G, lanes 1–3. G Same as A for BACE476Δ. H Same as B for BACE476Δ. I Same as A for NHK. L Same as B for NHK.