Schematic illustrating the in vitro expression cloning (IVEC) system used to identify and isolate cDNAs from human adult brain library that encode Hrs UIM-interacting proteins.

The number of proteins in each category is expressed as the percentage of the total number of different proteins identified from the screen.

(A) HeLa cells were transfected with the indicated plasmids and treated with proteasome inhibitor MG132 for 8 hours before harvest. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody, followed by immunoblotting with anti-HA antibody to detect HA-tagged ubiquitin conjugated to Munc18-1 (upper panel). The blot was then re-probed with anti-Myc antibody to detect Myc-tagged Munc18-1 protein (lower panel). (B) In vivo ubiquitination of Hsc70 was analyzed using the same assay as described above. Data are representative of at least 3 independent experiments.

(A) Domain structure of GST-Hrs fusion proteins. (B) Soluble immunopurified Myc-tagged Munc18-1 (input) was incubated with similar amounts of immobilized GST or GST-Hrs fusion proteins (lower panel). Immunoblot analysis of bound proteins with anti-ubiquitin antibody (upper panel) and anti-Myc antibody (middle panel) reveals an UIM-dependent interaction of Hrs with ubiquitinated Munc18-1 but not with non-ubiquitinated Munc18-1 protein. (C) Soluble immunopurified Myc-tagged Hsc70 (input) was incubated with similar amounts of immobilized GST or GST-Hrs fusion proteins (lower panel). Immunoblot analysis of bound proteins with anti-ubiquitin antibody (upper panel) and anti-Myc antibody (middle panel) reveals that Hrs binds ubiquitinated Hsc70 and non-ubiquitinated Hsc70 protein through different domains. The immunopurified Myc-tagged, ubiquitinated and non-ubiquitinated forms of Munc18-1 or Hsc70 in the input lane were detected by immunoblotting with anti-ubiquitin antibody and anti-Myc antibody, respectively, but their amounts were too low for detection by the Coomassie stain.

(A) Equal amounts of proteins from HeLa cell lysates transfected with the indicated siRNA were analyzed by immunoblotting with antibodies against Hsc70 and EEA1. (B) HeLa cells transfected with the indicated siRNAs were incubated with ¹²⁵I-EGF for 10 minutes at 37°C. The internalized ¹²⁵I-EGF is expressed as a percentage of the initially bound ¹²⁵I-EGF. (C) HeLa cells transfected with the indicated siRNAs were allowed to internalize ¹²⁵I-EGF for 10 minutes and then chased for 1 hour at 37°C. The degraded 125I-EGF is expressed as a percentage of the initially internalized ¹²⁵I-EGF. Data represent mean ± s.e.m. from 3 independent experiments. The asterisks indicate a statistically significant difference (p < 0.05) from the control siRNA-transfected cells.

(A) Domain structure of full-length Hrs (top) and the GST-fused Hrs UIM domain used in the IVEC screen (bottom). (B) Two cDNA pools, IA1 and IB1, containing 100 independent cDNA clones per pool from a human adult brain cDNA library, were in vitro transcribed and translated in the presence of cold methionine with or without ubiquitin. The control (CTL) reactions were carried out under the same conditions with no cDNAs added. The synthesized protein pools were analyzed by immunoblotting with anti-ubiquitin antibody. (C) Primary screen for positive pools containing Hrs UIM-binding proteins. Pools of cDNAs (100 independent cDNA clones per pool) from a human adult brain cDNA library were in vitro transcribed and translated in the presence of [³⁵S]methionine and ubiquitin and then subjected to a GST-Hrs UIM pull down assay. Bound proteins were analyzed by SDS-PAGE. Autoradiography of gel samples was performed using a phosphoimager. Example of positive pools (Pool # 1, 2, 4, 5, 7, and 8) selected for secondary screen. The two bands labeled a and b in Pool # 4 represent distinct Hrs UIM-binding proteins, which would be individually isolated by secondary screen. (D) Secondary screen for isolation of individual positive cDNA clones encoding Hrs UIM-binding proteins. In vitro translated products from individual cDNA clones isolated from each of the positive pools were analyzed as described above for their ability to bind GST-Hrs UIM. Example of 8 single clones isolated from Pool # 4, of which Clone # 3 and 5 are individual positive cDNA clones encoding Hrs UIM-binding proteins a and b indicated in (C). (E) Specificity of Hrs UIM binding. In vitro translated products from 3 isolated individual cDNA clones (Input) were incubated with immobilized GST-Hrs UIM fusion protein or GST control. Bound proteins were analyzed by SDS-PAGE and autoradiography. Clone # 2 encodes a protein that specifically binds to GST-Hrs UIM but not to GST control, whereas Clone # 1 and 3 are negative interactors that binds neither to GST-Hrs UIM nor to GST control.