Chen X , Tukachinsky H , Huang CH , Jao C , Chu YR , Tang HY , Mueller B , Schulman S , Rapoport TA , Salic A .

GM052586 NIGMS NIH HHS , Howard Hughes Medical Institute , R01 GM052586-18 NIGMS NIH HHS , R01 GM052586 NIGMS NIH HHS , R01 GM092924 NIGMS NIH HHS , T32 GM007753 NIGMS NIH HHS

	Figure 1. Processing of the purified Hh precursor. (A) A fusion was generated between maltose-binding protein (MBP), the last 15 amino acids of the N-terminal fragment of Drosophila Hh (DHh), and the entire C-terminal fragment of DHh (MBP-DHh). The purified protein was incubated for 5 h at room temperature with different concentrations of DTT in the absence or presence of cholesterol (Cho). The samples were analyzed by nonreducing SDS-PAGE and Coomassie staining. MBP-DHh–N and DHh-C are the N- and C-terminal fragments generated by Hh processing. (B) As in A, but the reaction contained 3H-labeled cholesterol. The samples were analyzed by reducing SDS-PAGE followed by either Coomassie staining or fluorography. (C) In vitro translated 35S-labeled wild-type (WT) MBP-DHh or the indicated cysteine mutants were incubated with 5-kD maleimide-polyethylene glycol (Mal-PEG) as indicated, in the presence or absence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). The samples were analyzed by reducing SDS-PAGE and autoradiography. The positions of singly and doubly Mal-PEG–modified species are indicated. The singly modified species have a different mobility, depending on which cysteine is modified. (D) As in A, but comparing wild-type MBP-DHh with the two cysteine mutants. Molecular masses are given in kilodaltons.
	Figure 2. Hh processing in Xenopus egg extracts. (A) The in vitro translated 35S-labeled Xenopus Sonic Hh (XShh) wild-type (WT) precursor was incubated at room temperature with Xenopus egg extracts for the indicated times. Parallel experiments were performed with three point mutants. The samples were analyzed by SDS-PAGE and autoradiography. The graph shows the quantification of the Hh precursor. (B) As in A, but the extract was supplemented with the indicated concentrations of oxidized or reduced glutathione (GSSG or GSH). (A and B) n = 3 time points. (C) In vitro translated 35S-labeled XShh fused at its N terminus to the maltose-binding protein (MBP-XShh) was incubated at room temperature with Xenopus egg extracts for the indicated times, in the absence or presence of 0.5% of either the cholesterol-sequestering detergents digitonin or cholate, or the control detergent Triton X-100 (TX-100). The samples were analyzed by SDS-PAGE and autoradiography. (D) In vitro translation was used to generate 35S-labeled fusions of MBP and either the N-terminal fragment of XShh (N), the C-terminal fragment of XShh (C), full-length XShh (FL), full-length XShh with a cysteine mutation in the active site (C199A), or XShh lacking the last 93 amino acids (ΔC). The fusions were incubated for 1 h with Xenopus egg extracts and subjected to Triton X-114 partitioning. Aliquots of the input (T), of the aqueous phase (A), or of the detergent phase (D) were analyzed by SDS-PAGE and autoradiography. (E) As in A, but with an MBP fusion of either wild-type XShh (FL) or a mutant lacking the last 93 amino acids (ΔC). Molecular masses are given in kilodaltons.
	Figure 3. Processing of HShh is dependent on disulfide bridge formation and reduction. (A) HShh-HA was stably expressed in 293T cells. Protein synthesis was inhibited with cycloheximide (CHX), and the fate of the protein was followed by SDS-PAGE and immunoblotting with anti-HA antibodies. Immunoblotting for p97 was used as a loading control. (B) As in A, but with HShh-HA containing a mutation in the catalytic cysteine (C198S). (C) As in A, but with HShh-HA containing a mutation in the conserved noncatalytic cysteine (C363A). (D) HA-tagged HShh was stably expressed in 293T cells. The cells were pulsed with [35S]methionine for 3 min and chase incubated with unlabeled methionine for the indicated times. 200 µM diamide or 0.5 mM DTT was added 10 min before the pulse and was present during the pulse and chase. The proteasome inhibitor epoxomicin (1 µM) was present, beginning at 1 h before the pulse. The samples were analyzed by immunoprecipitation with HA antibodies followed by reducing SDS-PAGE and fluorography. An equal number of cells were processed for each condition. (E) As in D, except that, where indicated, 10 µM brefeldin A and 1 µM epoxomicin were present, which were added 1 h before the pulse. The samples were analyzed as in D. Molecular masses are given in kilodaltons.
	Figure 4. PDI and PDIp are involved in the remodeling of the conserved disulfide bridge in HShh. (A) HShh-HA and FLAG-tagged Trx-like ER proteins, in which one of their CXXC motifs was changed to CXXA, were coexpressed in 293T cells. Cell extracts were subjected to immunoprecipitation (IP) with HA or FLAG antibodies followed by SDS-PAGE and immunoblotting (IB) with FLAG and HA antibodies. Where indicated, the immunoprecipitated samples were reduced with DTT before electrophoresis. (B) Wild-type (WT) HShh-HA or the processing-defective C198A mutant was coexpressed with a FLAG-tagged CXXA mutant of PDI (C56A) in 293T cells. The cells were pulse labeled with [35S]methionine for 3 min and chase incubated for different time periods. The proteasome inhibitor epoxomicin (1 µM) was added 1 h before the pulse. All samples were subjected to immunoprecipitation with HA antibodies followed by SDS-PAGE and fluorography. Where indicated, the samples were reduced with DTT before electrophoresis. Molecular masses are given in kilodaltons.
	Figure 5. HShh-C is not secreted and is degraded in the ER. (A) HShh-HA was stably expressed in 293T cells. The cells were washed and incubated for 12 h with DME containing 0.5% fetal bovine serum. The cell pellets and equivalent amounts of culture medium were analyzed for the presence of HShh-N and HShh-C by immunoblotting with HShh-N antibodies and HA antibodies. Where indicated, the proteasome inhibitor epoxomicin was present during the last 3 h of incubation. The graph shows the distribution of HShh-N and HShh-C between cells and medium. P, pellet. S, supernatant. Molecular masses are given in kilodaltons. (B) HShh was tagged with mCherry at its C terminus and stably expressed in 293T or in NIH-3T3 cells. Its localization was determined by fluorescence microscopy. The ER was revealed by immunostaining with rabbit antibodies against calnexin. The bottom row shows merged images. Bar, 20 µm. (C) Wild-type HShh-HA or the processing-defective mutant HShh-C198A-HA was stably expressed in NIH-3T3 cells. Cells were immunostained with rat HA and rabbit HShh-N antibodies followed by goat anti–rat Alexa Fluor 488 (green) and goat anti–rabbit Alexa Fluor 594 (red) secondary antibodies. The cells were incubated for 3 h with or without the proteasome inhibitor epoxomicin (1 µM). The third row shows merged images of the green and red channels. The bottom row shows differential interference contrast (DIC) images. Bar, 50 µm.
	Figure 6. ERAD components required for the degradation of HShh-C. (A) Cells were depleted of the ER luminal lectin OS9 by siRNA, and the fate of stably expressed HShh-HA was followed after cycloheximide (CHX) addition. The extent of OS9 depletion (in parentheses) was determined by quantitative RT-PCR. Controls were treated with an unrelated siRNA. All samples were analyzed by SDS-PAGE and immunoblotting with HA antibodies. The right graph shows quantification of HShh-C in the experiment. All samples were also analyzed by immunoblotting for p97 (loading control). (B) As in A, but with depletion of the ER luminal lectin XTP3 by two different siRNAs. (C) As in A, but with depletion of the ubiquitin ligase Hrd1 by two different siRNAs. (D) As in A, but with depletion of the Hrd1-interacting protein Sel1 by two different siRNAs. (A–D) n = 3 time points. (E) As in A, but with depletion of the ATPase p97 by siRNA. n = 4 time points. Molecular masses are given in kilodaltons.
	Figure 7. Dominant-negative ERAD components inhibit HShh-C degradation. (A) Cells stably expressing the HShh-HA precursor were transfected with a catalytically inactive Myc-tagged Hrd1 (Hrd1-C291A) or with an empty vector. The fate of HShh-HA was followed after addition of cycloheximide (CHX), by SDS-PAGE and immunoblotting for HA; immunoblotting for p97 served as a loading control. All samples were also analyzed by SDS-PAGE followed by immunoblotting for Myc (Hrd1-C291A) and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control). The right graph shows the quantification of the HShh-C in the experiment. n = 4 time points. (B) As in A, but with transfection of either wild-type p97 (p97-WT), a catalytically inactive p97 mutant (p97-QQ), or with an empty vector. The HA blot was stripped and reprobed with anti-GAPDH antibodies (loading control). Endogenous and overexpressed p97 were detected on the same gel by immunoblotting with anti-p97 antibodies. n = 3 time points. (C) HShh-HA was transiently expressed in 293T cells together with dominant-negative Ubc6e (Ubc6e-C91S), control vector, or UbxD8-GFP. Greater than 90% of the cells showed a strong GFP signal by live-cell fluorescence microscopy (not depicted). Protein synthesis was inhibited with cycloheximide, and the fate of HShh-HA was followed by SDS-PAGE and immunoblotting with HA antibodies. Ponceau S staining of the blot is shown to demonstrate the loading of equal amounts of protein. Molecular masses are given in kilodaltons.
	Figure 8. Cytoplasmic events preceding HShh-C proteolysis. (A) To test for deglycosylation of HShh-C, HShh-HA was stably expressed in 293T cells. A cycloheximide chase was performed for 2 h in the presence or absence of the proteasome inhibitor MG132. Cell lysates were incubated in the absence or presence of protein N-glycanase F (PNGase F) as indicated. Samples were analyzed by SDS-PAGE and immunoblotting with HA antibodies. Immunoblotting with p97 antibodies served as a loading control. (B) To test for polyubiquitination of HShh, cells stably expressing HShh-HA were incubated in the absence or presence of MG132 for 2 h. Extracts were subjected to immunoprecipitation (IP) with HA antibodies, and the proteins were analyzed by SDS-PAGE and immunoblotting (IB) with HA-antibodies (lanes 3 and 4) or ubiquitin (Ub) antibodies (lanes 1 and 2). Lanes 5 and 6 show blots of the extract before immunoprecipitation. (C) To test whether Hrd1 polyubiquitinates HShh, an experiment as in B was performed except that, where indicated, cells were transfected with a Myc-tagged dominant-negative Hrd1 mutant (Myc-Hrd1-291A). To test for the presence of the Hrd1 mutant, the samples were also analyzed by blotting with Myc antibodies. Immunoblotting for p97 served as a loading control. (D) Cells stably expressing HShh-HA were transfected with Myc-tagged wild-type Hrd1 (Hrd1-WT) or catalytically inactive Hrd1 mutants (Hrd1 C291A or Hrd1 C291A-C307A). As a control, a Myc-tagged version of reticulon 4A was used. Cell extracts were either analyzed directly (lanes 1–4) or subjected to immunoprecipitation with HA antibodies (lanes 5–8). All samples were analyzed by SDS-PAGE and immunoblotting with HA or Myc antibodies. Molecular masses are given in kilodaltons.

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