Early Delivery of Misfolded PrP from ER to Lysosomes by Autophagy
Figure 1
Aberrant trafficking of PrP-T182A. (a) Western blot of whole cell lysates prepared from N2a cells transiently expressing either Wt- or Mut-PrP carrying the 3F4 epitope (i.e., PrP(3F4)) for 16 h, before and after digestion with Endoglycosidase H (Endo H), followed by probing with 3F4 mAb. Wt-PrP has two N-linked glycosylation sites at positions 180 and 196, producing un-, mono-, and diglycosylated PrP. The T182A mutation disrupts the N-X-T glycosylation consensus site, resulting in loss of diglycosylated PrP. The lower molecular weight fractions of Wt-PrP and the core-glycosylated fraction of Mut-PrP are sensitive to Endo H, whereas higher molecular weight, Golgi-modified glycoforms, is resistant. (b) Western blot of Wt- and Mut-PrP recovered from the media (top) or lysates (bottom) of COS-7 cells transiently expressing PrPs for 16 h and then incubated for 90 min at 4°C in OptiPro media in the absence (−) or presence of PIPLC (+). Wt-PrP was detected in the media of PIPLC treated cells, whereas Mut-PrP was not (top panels), although the expression of each was evident in cell lysate preps (bottom panels), as revealed by D13 F (ab) anti-mouse-PrP antibody. (c) Immunofluorescence confocal microscopy of N2a cells expressing either Wt- or Mut-PrP(3F4) (red), costained for giantin or PDI (green). In contrast to Wt-PrP, Mut-PrP is consistently absent from the plasma membrane although it does label the ER and Golgi, indicating limited trafficking to at least cis-Golgi. Scale bars = 12 μm. (d) Sucrose gradient subcellular fractionation of N2a cells stably expressing Wt- or Mut-PrP(3F4) shows that Mut-PrP is distributed primarily in heavier fractions that correspond with the ER marker calnexin, compared with Wt-PrP that cofractionates primarily with the Golgi marker GM130. PrP is detected with 3F4 mAb.
