Fig 1.
USP8 interacts with helices α4, α5 and α6 of CHMP1B.
A: Scheme of CHMP1B with six predicted α-helices (α1- α6) and cDNA constructs used in this study. The numbers indicate positions of amino acid residues. Asterisk (*) indicate the lysine residues in positions 42, 59, 87 and 90. B, C: HEK293T cells were co-transfected with GFP-CHMP1B and Flag-USP8 constructs, and cell lysates were immunoprecipitated (IP) with anti-GFP or anti-Flag antibodies. IPs were revealed by western blot (IB) using either anti-GFP or anti-Flag antibodies. In (C), whole cell lysates (WCL) were immunoblotted with anti-Flag, anti-CHMP1B or anti-Tub to reveal protein amounts. Note that expression of each protein CHMP1B or USP8 stabilizes the other partner. D: Representation of the Venus Fluorescent protein (VFP) fusion constructs carrying Myc or HA tag. VN: Venus Nter, VC: Venus Cter. The numbers indicate positions of amino acid residues in USP8 or CHMP1B. E: VFP fluorescence of fixed HeLa cells co-transfected with Myc-VN-USP8 and HA-VC-CHMP1B. Scale bar: 20 μm. In (E’), quantification of VFP spots was performed using HCS Studio software on Myc and HA positive cells only (i.e. in transfected cells). Vertical axis indicates Venus spots count per HA and Myc positive cell. Scatter dot plots represent one representative experiment out of three. Values are mean ± SD. *p<0.01; **p<0.05 (Student’s t-test). F: HeLa cells were co-transfected with Myc-VN-USP8 and HA-VC-CHMP1B as in E and immunostained with early (EEA1) or late (LAMP1) endosomes markers. In (F’), quantification of overlap between USP8-CHMP1B VFP spots and EEA1, or LAMP1 is represented as a fraction of total VFP fluorescence. Vertical axis indicates overlap area. Histogram represent one representative experiment out of three. Values are Mean ± S.E.M. ****p<0.0001 (Student’s t-test).
Fig 2.
CHMP1B ubiquitination is controlled by USP8.
A-C: Wild-type or mutated GFP-CHMP1B constructs were transfected into HEK293T cells jointly with HA-ubiquitin (HA-Ub). In (B), cells were co-transfected with shUsp8 and in (C), cells were co-transfected with Flag-USP8, Flag-USP8C748A or Flag-USP8S680A constructs. Immunoprecipitations (IP) were carried out with anti-GFP antibodies after strong denaturation of the lysate and proteins were analyzed by immunoblot (IB) using either anti-HA (Ub) or anti-GFP (CHMP1B) antibodies. Whole cell lysates (WCL) were immunoblotted with anti-HA to reveal transfected Ub-HA and, in (B), with anti-USP8 to reveal endogenous USP8, and in (C), with anti-Flag to reveal Flag-USP8 constructs. Total protein amount is shown. A’, B’, C’: Quantification by densitometry of blots in A, B or C, respectively. Normalized signals are expressed as a fold-change over CHMP1B wild-type basal ubiquitination levels. Histograms represent the mean of two to three independent experiments. Error bars indicate the range. Values are mean ± SD. *p<0.05 (Student’s t-test).
Fig 3.
Polymeric CHMP1B belongs to a CHMP1B IST1 complex and is free of ubiquitin.
A: HEK293T whole cell lysate was analyzed by immunoblot with anti-CHMP1B antibody. B: HEK293T whole cell lysates were separated by sucrose gradient centrifugation. The different fractions were separated by SDS-PAGE and CHMP1B was visualized by immunoblot (IB). C: The 30% sucrose gradient fraction (in B) was concentrated and separated on a Superdex 200 column. 0.5 ml fractions were analyzed by IB against CHMP1B or IST1. The elution volume of marker proteins are indicated; thyroglobulin, 669 kDa, elutes at 9.8 ml; γ-globulin, 158 kDa, elutes at 13.2 ml. D: HEK293T whole cell lysate was immunoprecipitated with the FK2 (left) or the anti-CHMP1B antibody (right) as indicated on the top. The whole cell lysate (WCL), the unbound fraction (UF) and the IPs were analyzed by IB against CHMP1B. Asterisk indicates unspecific band (see S3C Fig). E: Schematic representation of the recognition site of the antibody against CHMP1B. F: HEK293T cells were transiently transfected with control (pSilencer) or shUsp8 plasmids for 48 hours. The whole cell lysate was immunoprecipitated with the FK2 antibody and the IPs were analyzed by IB against CHMP1B. Asterisk indicates unspecific band at 35 kDa (see S3C Fig). Double Asterisk corresponds to light and heavy chains of IgG (at 25kDa and 55 kDa, respectively).
Fig 4.
Dynamic ubiquitination of CHMP1B in response to EGF stimulation.
A: GFP-CHMP1B constructs were transfected into HEK293T cells jointly with HA-ubiquitin (HA-Ub). Cells were starved for 16 hours prior to stimulation by EGF at 100 ng/ml. Immunoprecipitations (IP) were carried out with anti-GFP antibodies of the lysate and proteins were analyzed by immunoblot (IB) using either anti-HA (Ub) or anti-GFP antibodies. Whole cell lysates (WCL) were immunoblotted with indicated antibodies. B: Serum starved HEK293T cells were subjected to EGF at 100ng/ml stimulation. Whole cell lysates (Whole) and cell fractions (Cytosolic and Membrane) were prepared as indicated in supplemental methods. Briefly, cell debris and nuclei were eliminated though a brief centrifugation and cell lysates were then re-centrifuged at 13000 x g for 20 min at 4°C. The supernatant was collected as cytosolic fraction while the pellet was re-suspended, briefly centrifuged to eliminate insoluble proteins, and collected as membrane fraction. Ubiquitinated proteins were immunoprecipitated with FK2 antibody from either the WCL (1), the cytosolic (2) or the membrane (3) fractions at the indicated time of EGF stimulation. IPs were analyzed by SDS-PAGE and revealed by IB using anti-CHMP1B or FK2 antibodies. Total proteins are shown. Lamp1 expression was analyzed from supernatant or pellet before IP.
Fig 5.
Ubiquitination of CHMP1B is required for EGFR degradation.
A: Analysis of lysates from HeLa cells expressing or not a shRNA-CHMP1B against the 3’UTR region (sh94) and transfected with HA-empty, HA-CHMP1B-WT or HA-CHMP1B-4K>R as indicated. Cells were subjected to 16 hours serum starvation prior to stimulation by EGF and lysed at the indicated time points prior to analysis. Proteins were separated by SDS-PAGE and revealed by immunoblots (IB) using either anti-EGFR or anti-HA antibodies. Total protein amounts are shown. A’: Quantification of the amount of EGFR was carried out by densitometry and normalized signals are expressed as a fold-change over the basal EGFR level at time 0 minutes (in the absence of EGF) to circumvent variations in total EGFR amount prior to stimulation. Histograms represent the mean of three independent experiments. Error bars indicate standard deviation. Statistical significance was determined using two-way ANOVA (***p<0.001, **p<0.001).
B: Confocal images of control or CHMP1B-silenced (shCHMP1B-94) HeLa cells transfected with HA-empty, HA-CHMP1B or HA-CHMP1B-4K>R. Cells were subjected to serum starvation prior to stimulation by EGF and fixed at the indicated time points. Immunofluorescence staining of non-permeabilized cells was performed to stain specifically plasma membrane EGFR. Scale bar: 40μm. B’: Quantification of the intensity of fluorescence per cell was performed using Cell Profiler Software. Vertical axis indicates fluorescence intensity units per cell. Histograms represent the mean intensity measured from individual cells. Error bars indicate standard deviation. The numbers between parentheses indicate the number of cells measured for each experimental condition. Statistical significance was determined using two-way ANOVA (****p<0.0001). Note that CHMP1B-WT expression in shCHMP1B context induced an apparent complete rescue of EGFR disappearance from the cell membrane while the immunostaining revealed only about 50% of CHMP1B-WT expressing cells (as shown in S5B Fig). One possible explanation is that a number of transfected cells express low levels of the rescuing CHMP1B protein that are not detectable by immunofluorescence but sufficient for the rescue of CHMP1B knock-down. In addition, overexpression of CHMP1B reduced the level of EGFR at the membrane compared to control cells as clearly observed at the starting point (see time 0) which may also account for the apparent total rescue of the phenotype. C: Confocal images of HeLa cells or CHMP1B-silenced HeLa cells (shCHMP1B-94). Cells were subjected to 16 hours serum starvation prior to stimulation by EGF and fixed at 30 min of stimulation. Immunofluorescence staining was performed on permeabilized cells using anti-EGFR and anti-RAB4 antibodies. Scale bar: 20μm. C’: Left histogram: EGFR and RAB4 dots were segmented and their intensity measured using the Cell Profiler software. The mean intensity and the standard deviation (error bars) calculated from individual dots are shown. The numbers between parentheses indicate the number of dots measured for each experimental condition. Statistical significance was determined using T-test (****p<0.0001). Right histogram: the Pearson coefficient correlation between EGFR and RAB4 dots was calculated for each cell using the Cell Profiler software. The mean Pearson coefficient and the standard deviation (error bars) calculated from individual cells are shown. The numbers between parentheses indicate the number of cells measured for each experimental condition. Statistical significance was determined using T-test (****p<0.0001). D, D’: Control (shNT) and shCHMP1B-94 silenced HeLa cells (shCHMP1B) were serum starved for 16 hours. An antibody directed against the extracellular domain of EGFR was added in the culture medium together with EGF and the EGFR/antibody complex was let allowed to internalize for the indicated time of EGF stimulation before fixation. D: Cells stained with EGFR were imaged by automated ArrayScan microscope. D’: Quantification of EGFR spots was performed using the dedicated software (see methods). Scale bar: 40μm. Statistical significance was determined using two-way ANOVA (****p<0.0001).
Fig 6.
The ubiquitin deficient form of CHMP1B is not functional in Drosophila.
A: Protein sequence alignment of Drosophila melanogaster CHMP1 versus its Homo sapiens orthologue CHMP1B. Lysine residues in Hs-CHMP1B are marked in yellow. Conserved lysine residues in Dmel-CHMP1 are marked in green, the one which is not is marked in red. The four lysine substituted by arginine residues in CHMP1B-4K>R are marked with red arrow-heads. The MIM (MIT interacting domain) is marked in grey. B: Pictures of 3 to 5 day-old female flies raised at 25°C expressing the indicated transgenes under the control of the wing MS1096-Gal4 driver. Genotypes are as follows in w1118 background. Ctrl: MS1096/+. Chmp1-IR: MS1096/+;UAS-lacZ/+;UAS-Chmp1-IR/+. Usp8-IR: MS1096/+;UAS-Usp8-IR/+;UAS-birA. The number of flies examined is N>100 in all cases. The phenotypes were 100% penetrant in all cases. C: Upper panels: confocal images of dissected late third instar larval wing imaginal discs. EGFP is expressed under the control of Notch Responding Elements (NRE). Myr-RFP is expressed under the control of UAS sequences. Gal4 is expressed under the control of engrailed (en) promoter. a,b,c,d: frontal average Z stack of confocal series. a’ and a” show lateral views of the Z stack indicated by white arrowheads in (a), at the level of either the non-Gal4 expressing area (a’, corresponding to anterior part of the wing imaginal disc) or the Gal4 expressing area (a”, corresponding to the posterior part of the wing imaginal disc). Same configuration for b’, b”, c’, c”, d’, d”. Ap: apical side. Bs: basal side. Magnification objective 63x. Scale bar: 75μm. Bottom panels: pictures of corresponding adult wings. The penetrance of the phenotype is indicated in percentage calculated on (N) flies examined as indicated. Genotypes are as follows: Control: w1118; en-Gal4, UAS-Myr-RFP, NRE-EGFP/+. Chmp1-IR: w1118; en-Gal4, UAS-Myr-RFP, NRE-EGFP/+; UAS-Chmp1-IR/+. Chmp1-IR + hCHMP1B: w1118; en-Gal4, UAS-Myr-RFP, NRE-EGFP/UAS-hCHMP1B; UAS-Chmp1-IR/+. Chmp1-IR + hCHMP1B-4K>R: w1118; en-Gal4, UAS-Myr-RFP, NRE-EGFP/UAS-hCHMP1B-4K>R; UAS-Chmp1-IR/+.