FIG. 4.

Repression of SEC39 alters Pex3p trafficking. (A) Equal amounts of protein from whole-cell lysates prepared from THC-SEC39 cells containing genomically integrated PEX3-GFP under the control of the GAL1 promoter at the PEX3 locus were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted for GFP, Sec39p, and Gsp1p as a loading control. (B) THC-SEC39 cells integrated for pGAL1-PEX3-GFP were fixed with formaldehyde prior to image capture by epifluorescence microscopy. Arrows indicate the accumulation of Pex3p-GFP in tubular-vesicular structures with apparent perinuclear localization. Prime, cells were cultured for 8 h in YEPR to derepress the GAL1 promoter for subsequent galactose induction of PEX3-GFP expression. Pulse, cells were then transferred to YEPG and incubated for 1 h to pulse-label the cells for PEX3-GFP expression. Chase, expression of PEX3-GFP was then stopped by changing the growth medium to YEP2×D, and cells were incubated for an additional 4 h and 15 h to monitor the trafficking of Pex3p-GFP between the ER and peroxisomes. Repression of SEC39 was regulated by the addition or omission of 10 μg doxycycline/ml to the culture medium. Bar, 5 μm. (C) THC-SEC39-pGAL1-PEX3-GFP, and THC-SEC61-pGAL1-PEX3-GFP cells were subjected to prime, pulse, and chase incubation steps as described for panel B, except that the prime step was for 18 h and the chase step was for 4 h. Cells were fixed in formaldehyde prior to image capture by epifluorescence microscopy at the indicated steps. Bars, 5 μm. (D) Equal amounts of protein from whole-cell lysates of the THC-SEC39-pGAL1-PEX3-GFP and THC-SEC61-pGAL1-PEX3-GFP cells used for panel C were resolved by SDS-PAGE, transferred to nitrocellulose and immunoblotted for GFP, Sec39p, Sec61p, Gsp1p, and Act1p. Gsp1p and Act1p served as loading controls.