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. 2002 Jul 8;158(1):91–102. doi: 10.1083/jcb.200201053

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Copyright © 2002, The Rockefeller University Press

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Figure 6. — Rsp5p is the ubiquitin ligase of the HIP pathway and is responsible for the ubiquitination of CPY* in HRD/DER pathway–deficient cells. (A) Ubiquitination of CPY* in wild-type (lane 1), prc1Δ hrd1Δ (lane 2), hrd1Δ (lane 3), rsp5–2 (lane 4), and hrd1Δ rsp5–2 (lane 5) cells was assayed by radiolabeling at 37°C followed by sequential immunoprecipitations first with anti-CPY antibodies and then with anti–ubiquitin-HA antibodies. (B and C) rsp5–2 (KHY355), hrd1Δ (KHY171), hrd1Δ rsp5–2 (KHY359), and wild-type (KHY163) strains were radiolabeled, chased (at 37°C), and CPY* immunoprecipitated at various times. Samples were analyzed as in Fig. 1. (B) Wild-type and rsp5–2 cells overexpressing (OE) CPY* (pAC453). (C) Wild-type, hrd1Δ, rsp5–2, and hrd1Δ rsp5–2 expressing single copy levels of CPY*. (D) Wild-type (RH448) and ubc4Δ ubc5Δ (RH3097) overexpressing (OE) CPY*–HA were radiolabeled (at 30°C), chased, and CPY* immunoprecipitated.