Figure 4.

Parkin interacts with, ubiquitinates and promotes the degradation of CDCrel-1. (A) Lysates prepared from HEK 293 cells transfected with pRK5-HA-CDCrel-1 and pRK5-myc-Parkin constructs were subjected to immunoprecipitation (IP) with anti-myc antibody followed by anti-HA immunoblotting. This experiment was replicated three times with similar results. (B) The ring-finger domains of Parkin interact with CDCrel-1. Lysates prepared from HEK 293 cells transfected with pRK5-HA-CDCrel-1 and myc-tagged Parkin domain constructs were subjected to immunoprecipitation (IP) with anti-myc antibody followed by anti-HA immunoblotting. This experiment was replicated three times with similar results. (C) Parkin ubiquitinates CDCrel-1. HEK 293 cells were transfected with pRK5-Myc-CDCrel-1, pMT123-HA-ubiquitin and pCMV-Parkin or control plasmid. Cells were then treated with 1 or 2 μM clasto-Lactacystin β-lactone for indicated times. Lysates were prepared from these cells and subjected to immunoprecipitation (IP) with anti-myc antibody followed by anti-HA immunoblotting. This experiment was replicated with similar results. (D) Parkin promotes the degradation of CDCrel-1. HEK 293 cells were transfected pRK5-myc-CDCrel-1 and pRK5-myc-Parkin or empty vector. 24 h after transfection, cells were washed and incubated with methionine-free medium for 1 h. The cells were pulsed-chased with 100 μCi of [³⁵S]methionine and were harvested at the indicated times for immunoprecipitation with anti-myc antibody. The immunoprecipitates were resolved on a 10% SDS/PAGE gel and visualized and quantitated with a phosphoimager. This experiment was replicated with similar results. (E) Familial-linked Parkin mutants fail to degrade CDCrel-1. The same experimental paradigm as described in Fig. 4D were performed with the familial-linked Parkin mutant pRK5-myc-Parkin-Thr415Asn or pRK5-myc-ParkinGln311 and compared with wild-type Parkin. This experiment was replicated with similar results.