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Vol. 18, Issue 10, 3952-3965, October 2007
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Hospital for Sick Children Research Institute, Department of Biochemistry and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, M5G 1X8
Submitted July 18, 2007;
Revised July 25, 2007;
Accepted July 31, 2007
Monitoring Editor: Sandra Schmid
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bonifacino and Traub, 2003; Hicke and Dunn, 2003). Although cell surface resident polypeptides can be transiently down-regulated by internalization, regulated degradation of receptors and transporters requires postendocytic delivery to lysosomes, a process that can be signaled by ubiquitination (Di Fiore et al., 2003; Dupre et al., 2004).
Conjugation of a single Ub (herein after designated mono-Ub) was proposed to be sufficient as an endocytic and vacuolar sorting signal in yeast and mammalian cells (Hicke, 2001; Mosesson et al., 2003). Recent evidence, however, suggests that multiple mono-Ub (designated multimeric-Ub) or Ub chains (designated polymeric-Ub or poly-Ub) are associated with efficient endocytic cargo recognition in animal cells (e.g., epidermal growth factor receptor [EGFR], major histocompatibility complex [MHC] I, and MHC II; Duncan et al., 2006; Huang et al., 2006; Ohmura-Hoshino et al., 2006; Shin et al., 2006; van Niel et al., 2006), as well as in yeast (e.g., Gap1 amino acid permease, Fur4p uracil permease; Springael et al., 1999; Dupre and Haguenauer-Tsapis, 2001; Soetens et al., 2001; Dupre et al., 2004).
After internalization, ubiquitinated cargo is recognized by the Ub-interacting motif (UIM) of hepatocyte growth factor–regulated tyrosine kinase phosphorylated substrate (Hrs) and STAM (Williams and Urbe, 2007), mammalian orthologues of the yeast Vps27 and Hse1, respectively (Hurley and Emr, 2006). These are conserved multidomain adaptors of ESCRT 0 (the endosomal sorting complex required for transport; Hurley and Emr, 2006; Williams and Urbe, 2007). Besides recognizing ubiquitinated cargo, the Hrs/STAM complex binds to PtdIns3P and clathrin heavy chain, facilitating clathrin recruitment to the limiting membrane of early endosomes, a major component of the endosomal double-layered coat (Raiborg et al., 2002). Recognition and concentration of ubiquitinated cargo by the Hrs/STAM complex at the double-layered coat is a prerequisite for cargo segregation and targeting to lysosomes (Raiborg et al., 2002). Successive cargo transfer from ESCRT0 to ESCRTI, II, and III coincides with endosomal maturation, manifesting in compositional changes, luminal acidification, and inward vesicular budding that leads to the formation of late endosomes enriched in multivesicular bodies (MVB; Mellman, 1996; Mukherjee et al., 1997). MVB formation ensures that cytosolic domains of transmembrane proteins become accessible to lysosomal proteases, a process promoted by endo-lysosomal fusions (Gruenberg and Stenmark, 2004; Hurley and Emr, 2006).
The essential role of poly-Ub or multimeric-Ub in rapid internalization of transmembrane proteins (e.g., CD4, 
2-adrenergic receptor complex, MHC I, MHC II, and ENaC; Traub and Lukacs, 2007) suggested that polyvalent interactions are required to overcome the low-affinity binding of Ub to the UIM of Ub-binding clathrin adaptors (e.g., epsin and eps15; Barriere et al., 2006; Hawryluk et al., 2006). Elucidating the significance of Ub configuration in lysosomal sorting is hampered by difficulties to discriminate between the role of Ub as an endocytic and lysosomal-sorting signal. Although previous observations suggest that short poly-Ub chains with various topologies may mediate lysosomal targeting of plasma membrane proteins (Staub et al., 1997; Geetha et al., 2005; Duncan et al., 2006; Kamsteeg et al., 2006; Wiemuth et al., 2007), the Ub configuration that is necessary and sufficient to signal lysosomal sorting has not been determined. Because the UIMs of epsin and eps15/eps15R, Ub-binding clathrin adaptors, as well as of Hrs/STAM appear to be critical for ubiquitinated cargo recognition, we hypothesized that similar principles may prevail for Ub recognition at the cell surface and endosomes.
To address the Ub configuration requirement for endolysosomal sorting, we used chimeric models and CD4, exposing mono-Ub, multimeric-Ub, or poly-Ub in their cytoplasmic tails. Interorganellar transport kinetics of cargoes with different Ub configurations were determined by vesicular pH measurements using fluorescence ratio image analysis (FRIA) of fluorophore-labeled cargoes. The results show that both multimeric and polymeric, but not monomeric Ub are efficiently recognized as lysosomal-targeting signal by the endosomal sorting machinery, consistent with the Ub-binding preference of Hrs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). HEK293T cells were stably transfected with the pIRES2-puro expression construct (BD Biosciences, San Jose, CA) and CD4Tl- or CD4TlUb-expressing clones were selected in the presence 10 µg/ml puromycin.
Transient transfection of COS-7, HEK293, and ts20/E36 cells was performed as described (Barriere et al., 2006). For transient cotransfection, the Ub variant and cargo cDNA was used at a 3:1 mass ratio.
DNA Constructs and Antibodies
CD4-Ub chimeras with the Lys-less linker (Tl) or the tetramerization coiled-coil domain (cc) from the potassium channel Kir6.2 have been described (Zerangue et al., 1999; Barriere et al., 2006). The terminal glycine residues in Ub were deleted by PCR mutagenesis (CD4Tl-UbR
G and CD4Tl-UbG) using the wild-type (wt) Ub and the Lys-less Ub (UbR) cDNA as template (provided by Dr. L. Hicke, Northwestern University, Evanston, IL). Lys residue was inserted into the UbRG moiety of CD4Tl-UbRG at the 6, 29, 48, or 63 position by overlapping PCR. In CD4-1K, the CD4 cytoplasmic tail (MSQIKRLLSEKKTCQCPHRFQKTCSPI) was substituted with RMSQIRRAASERKTCQCPHRFQ peptide to replace Lys436 and Lys442 residues with Arg and the di-Leu endocytosis motif with Ala, indicated by underscored letters. Thus the CD4-1K cytoplasmic tail contains a single Lys (at position 443) and lacks the last six-amino acid residues. All constructs were verified by DNA sequencing. HA-tagged Ub, UbR, UbR63K, and UbR48K (gift of Y. Yarden, Weizman Institute, Rehovot, Israel) have been described (Mosesson et al., 2003).
Glutathione S-transferase (GST) fusions, containing one (GST-Ub), two (GST-2Ub), and three Ubs (GST-3Ub) were described (Barriere et al., 2006). GST-Ub-L-Ub with a 10-amino acid linker between the Ubs and GST-Ub-Ub1A, containing UbI44A, were constructed by PCR mutagenesis. The GST-4Ub was generated by in frame insertion of PCR amplified Ub cDNA into the GST-3Ub. MBP-Hrs-His6 and MBP-HrsS270A-His6 were obtained by subcloning the mouse Hrs cDNA from pGEM-myc-Hrs and pGEM-myc-HrsS270A (provided by Dr. H. Stenmark, Norwegian Radium Hospital, Oslo, Norway) into pMAL-c2. The C-terminal His6 was introduced by PCR.
The following antibodies were used: anti-CD4: RPA-T4 (BD Biosciences, Oakville, ON, Canada), OKT4 (Harlan Bioproducts, Indianapolis, IN), rat monoclonal (Serotec, Raleigh, NC) and rabbit polyclonal (H-370, Santa Cruz Biotechnology, Santa Cruz, CA). The Lamp1 (H4A3) mAb has been developed by J. August (University of Iowa) and J. Hildreth (University of Iowa) and was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). The rabbit anti-Hrs was kindly provided by Dr. H. Stenmark. The anti-Ub is a monoclonal anti-Ub horseradish peroxidase (HRP)-conjugated (P4D1, sc-8017 Santa Cruz).
Vesicular pH Measurement
To monitor the endocytic trafficking of internalized chimeras, CD4, and other transmembrane proteins, the vesicular pH (pHv) of cargo-containing vesicles was determined by FRIA essentially as described for CFTR (cystic fibrosis transmembrane conductance regulator; Sharma et al., 2004). Cell surface CD4 (OKT4) and Lamp1 (Developmental Studies Hybridoma Bank) were labeled with the relevant primary Ab and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary Fab (Jackson ImmunoResearch Laboratories, West Grove, PA) by incubating primary (1/100) and secondary (1/100–500) Abs together routinely for 1 h at 37°C. Then cells were washed (140 mM NaCl, 5 mM KCl, 20 mM HEPES, 10 mM glucose, 0.1 mM CaCl2, and 1 mM MgCl2, pH 7.3) and chased for 30–90 min at 37°C. Fluid phase Ab uptake was not detectable by FRIA in mock-transfected cells (data not shown).
Lysosomes were labeled overnight in the presence of FITC-dextran or Oregon488-dextran (50 µg/ml, MW, 10 kDa, Molecular Probes, Eugene, OR) and chased >3 h (Poet et al., 2006) or loaded for 15 min with 5 mg/ml FITC-dextran and chased for 90 min. Recycling endosomes were labeled with FITC-transferrin (Tf; 5 µg/ml, 1 h labeling after 45-min serum depletion at 37°C) and chased for 10 min. FITC-Fab antibody, FITC-Tf, FITC-dextran, and FITC/Oregon488-loaded endolysosomes were imaged on an Axiovert 100 inverted fluorescence microscope (Carl Zeiss MicroImaging, Toronto, ON, Canada) at 35°C, equipped with a Hamamatsu ORCA-ER 1394 (Hamamatsu, Japan) cooled CCD camera and a Planachromat (63x, NA 1.4) objective. Fluorescence ratio image acquisition and analysis were performed with the MetaFluor software (Molecular Devices, Downingtown, PA). Images were acquired at 490 ± 5- and 440 ± 10-nm excitation wavelengths, using a 535 ± 25-nm emission filter.
Calibration curves, describing the relationship between the fluorescence ratio values and pHv served to calculate the luminal pH of individual vesicles after fluorescence background subtraction at both excitation wavelengths. In situ calibration was performed by clamping the pHv between 4.5 and 7.4 in K+-rich medium (135 mM KCl, 10 mM NaCl, 20 mM HEPES or 20 mM MES, 1 mM MgCl2, and 0.1 mM CaCl2) in the presence of 10 µM nigericin and 10 µM monensin (Sigma-Aldrich, Oakville, ON, Canada) and recording the fluorescence ratio. In each experiment the pHv of 200–800 vesicles was determined, precluding those located at the subplasma-membrane region. To ensure that comparable number of vesicles was evaluated for each constructs, three times more cells were analyzed for chimeras with high recycling efficiency (e.g., CD4Tl and CD4Tl-UbRG). As an internal control, one point calibration was performed on each coverslip by clamping the pHv to 6.5 with monensin and nigericin. Mono- or multipeak Gaussian distributions of pHv values were obtained with Origin 7.0 software (OriginLab, Northampton, MA). The mean pHv of each vesicle population was calculated because the arithmetic mean of the data and was identical to the Gaussian mean, based on single-peak distribution fitting. At least three independent experiments were performed for each condition. HEK293T cells constitutively expressing CD4Tl-Ub were overtransfected with Ub variants and the expression plasmid encoding the dsRed fluorescent proteins (BD Biosciences) at 10:1 ratio. FRIA was performed on cells expressing the dsRed only. The mean pHv obtained in three or more experiments is shown in Supplementary Table S1.
To confirm that the primary and secondary Ab remains bound to CD4 in late endosomes, the pH resistance of Ab binding was measured. After primary and HRP-conjugated secondary Ab binding to CD4Tl-expressing HEK293 cell at 0°C the pH of the extracellular medium was changed to pH 7.2, 5.0, and 2.5 for 5 min. HRP-conjugated Ab binding was measured by fluorescence, using Amplex-Red as substrate (data not shown). The Ab binding was virtually unaltered at pH 5.0, but was reduced by 95% at pH 2.5.
Considering that the pH sensitivity of FITC is reduced at pH <5 (pKa 
6.4), we validated our pHv measurements at pHv <5. To this end the lysosomal compartment was loaded with a mixture of Oregon-Green (75%) and FITC-dextran (25%) to increase the detection sensitivity at acidic pH, because the Oregon-Green pKa is 4.7. FRIA showed that Oregon-Green/FITC-dextran loading provided pH values similar to that obtaining in the presence of FITC alone, verifying the extrapolation procedure for pHv <5 based on FITC loading alone (data not shown). All experiments were performed in COS-7, and critical ones were repeated in HEK293 cells with similar results.
Immunofluorescence Microscopy
Lysosomes were labeled with anti-Lamp1 Ab or by FITC-dextran (50 µg/ml, MW, 10 kDa), with loading as described for pHv determination. Cells expressing the chimera were allowed to internalize CD4 antibody (RPA-T4, BD Biosciences) complexed with FITC-conjugated goat anti-mouse Fab or TRITC-conjugated goat anti-mouse IgG for 0.75–1 h in DMEM and chased for 0.5 h. During the last 45 min, cells were labeled with FITC-Tf or TRITC-Tf (5 µg/ml) after 45-min serum depletion to visualize recycling endosomes. Single optical sections were collected by Zeiss LSM510 laser confocal fluorescence microscope, equipped with a Plan-Apochromat 63x/1.4 (Carl Zeiss Microimaging) as described (Lechardeur et al., 2004). Images were processed with Adobe Photoshop (Adobe Systems, San Jose, CA) software.
Recombinant Protein Purification and Pulldown Assay
GST-Ub fusions proteins were purified essentially as described (Barriere et al., 2006). Expression of His6-tagged MBP-HrsS270A, MBP-Hrs298X, and MBP was induced in HB101 cells with 0.3 mM IPTG (4 h, RT). MBP-Hrs-His6 was expressed in the BL21(DE3) strain. For the Hrs pulldown experiments, the bacterial expression level of recombinant MBP-Hrs proteins was determined by affinity purification using amylose beads. Next, 65 pmol of fusion proteins was bound to amylose beads (overnight, 4°C), washed three times (20 mM Tris-Cl, pH 7.4, 200 mM NaCl, 1 mM EDTA), and incubated with mono-Ub (15 µg, Sigma), K48- (15 µg), or K63-linked (6 µg) poly-Ub chain (Boston Biochem, Cambridge, MA) for 2 h at 4°C. Bound proteins were eluted with 2x LSB. Six percent of K63- and 16% of K48-linked poly-Ub and mono-Ub present in the binding buffer were loaded directly to compensate for the approximately twofold lower detection efficiency of K48 poly-Ub by the P4D1 anti-Ub Ab.
Cell Surface Density, Internalization, Recycling, and Stability of CD4-Ub Chimeras
The cell surface density and internalization of CD4-Ub chimeras was measured essentially as described previously (Barriere et al., 2006), using anti-CD4 Ab. The remaining anti-CD4 Ab was detected by HRP-conjugated goat anti-mouse Ab and AmplexRed as a fluorescent substrate (Molecular Probes). Fluorescence was measured by OPTIMAstar (BMG Labtech, Offenburg, Germany) plate reader.
Recycling of chimeras was monitored by the biotin-streptavidin sandwich techniques as described (Sharma et al., 2004) with the modification that endosomes were loaded for 30 min with anti-CD4 and biotinylated anti-mouse Abs, and then the cell surface–resident biotinylated secondary Ab was blocked with 20 µg/ml streptavidin (Sigma-Aldrich; 4°C, 1 h). Exocytosis of internalized cargo was stimulated by shifting the temperature to 37°C for 5–10 min and detected by HRP-conjugated streptavidin and AmplexRed. Recycling efficiency was expressed as % of endocytosed cargo, measured in parallel samples.
The turnover of plasma membrane–associated CD4 chimeras was monitored by the disappearance kinetics of cell surface–bound anti-CD4 Ab during 0.5–20-h chase at 37°C in transiently transfected COS-7 cells. The cell surface bound anti-CD4 Ab was determined HRP-conjugated secondary Ab, using Amplex-Red as substrate.
Immunoprecipitation and Western Blotting
To determine the endogenous ubiquitination level of various CD4-chimereas at the cell surface and endosomes, the chimeras were labeled with anti-CD4 (OKT4) Ab for 30 min at 37°C in transiently transfected COS-7 cells. To delay degradation of the ubiquitinated chimeras the medium was supplemented with 20 µg/ml leupeptin, 20 µg/ml pepstatin, and 1 µM bafilomycin A1. Cells were lysed with RIPA buffer containing 10 µg/ml leupeptin and pepstatin, 100 µM phenylmethylsulfonyl fluoride, 10 µM MG132, and 10 mM NEM. Anti-CD4 Ab complexes were isolated on protein G agarose (Invitrogen, Carlsbad, CA; 2 h, 4°C), bound proteins were eluted with 2x LSB and probed with polyclonal anti-CD4 (H-370, Santa Cruz Biotechnology) or monoclonal anti-Ub HRP-conjugated (P4D1, sc-8017; Santa Cruz Biotechnology). To monitor the incorporation of heterologous Ub, CD4Tl-Ub–expressing HEK293 cells were transiently transfected with HA-tagged Ubwt, UbR, UbR63K, or UbR48K. After 48 h, cells were lysed in RIPA buffer as above. CD4Tl-Ub was precipitated with anti-CD4 rat monoclonal Ab (Serotec) and protein G agarose beads (Invitrogen). Bound proteins were probe with polyclonal anti-CD4 (H-370; Santa Cruz Biotechnology) or anti-HA Ab (Covance; MMS-101R).
Densitometric analysis of immunoblots was performed with the NIH Image 1.62 software as described (Sharma et al., 2004). Immunoprecipitations of Hrs with CD4Tl-Ub was carried out as outlined previously for CFTR (Sharma et al., 2004) with the following modifications. HEK293 cells expressing CD4Tl-Ub were incubated with MG132 (10 µM, 1 h) and lysed 150 mM NaCl, 20 mM Tris-Cl, and 0.2% NP-40, pH 7.4, containing 10 µg/ml leupeptin and pepstatin, and 0.1 mM phenylmethylsulfonyl fluoride, 10 µM MG132, and 10 mM NEM. The chimera was precipitated with the OKT4 Ab.
Statistical Analysis
Experiments were repeated at least three times or as indicated. Data are means ± SEM. Significance was calculated by using the two-tailed p value at 95% confidence level with unpaired t test, using the Prism software (GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Pitcher et al., 1999). The truncated CD4, consisting of the extracellular and transmembrane domain of CD4 (CD4Tl) is targeted to the plasma membrane by default and lacks any sorting signal (Bedinger et al., 1988; Barriere et al., 2006). We engineered three CD4-Ub chimeras, exposing either a poly-Ub chain (CD4Tl-Ub), an unextendable mono-Ub (CD4TI-UbRG), or a tetrameric unextendable Ub (CD4cc-UbRG) as their cytoplasmic sorting signal to assess the structural basis of Ub-dependent lysosomal sorting of transmembrane cargo (Figure 1a).
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G, Figure 1, a and b). Deletions of Gly residues prevented the possible recruitment of the ubiquitination machinery (Reyes-Turcu et al., 2006). Finally, to mimic multiple monoubiquitination (Haglund et al., 2003), the cytoplasmic linker of CD4Tl-UbRG was substituted with the cc tetramerization domain of the Kir6.2 channel (CD4cc-UbRG; Yuan et al., 2003). This tetramerization domain provoked the oligomerization of soluble and chimeric proteins (Barriere et al., 2006) and was postulated to expose tetrameric, unextendable Ub.
To demonstrate the differential ubiquitination of the three model proteins, the cell surface resident and internalized CD4-Ub chimeras were immunoisolated after anti-CD4 antibody binding and endocytosis, as described in Materials and Methods. This approach precluded the detection of ubiquitinated chimera associated with the endoplasmic reticulum (ER) and Golgi compartments. Probing the immunoprecipitates with anti-Ub showed that CD4Tl-Ub is subjected to polyubiquitination. In contrast, we could not to detect Ub conjugation to CD4Tl-UbRG and CD4cc-UbRG (Figure 1b), supporting the prediction that these fusion proteins expose a single, covalently attached Ub in vivo.
Poly-Ub and Multimeric-Ub Metabolically Destabilizes CD4 Reporter Proteins
The metabolic stability of chimeras was measured by immunoblotting after the inhibition of protein synthesis with cycloheximide (CHX). Both CD4Tl-Ub and CD4cc-UbRG have significantly faster turnover rates than either CD4Tl or CD4Tl-UbRG (Figure 1, c and d). The accelerated degradation of CD4Tl-Ub and CD4cc-UbRG, at least in part, can be attributed to lysosomal proteolysis, because preventing lysosomal acidification and delivery by bafilomycin A1, a vacuolar H+-ATPase inhibitor, or blocking lysosomal cathepsins by leupeptin/pepstatin, significantly delayed the degradation (Figure 1, e and f; van Weert et al., 1995; Aniento et al., 1996). The cysteine protease and proteasomal inhibitors MG132 and lactacystin also stabilized the chimeras, conceivably by depleting endogenous Ub and/or interfering with cargo budding into MVB, as documented for ubiquitinated EGF and PDGF (platelet-derived growth factor) receptors (Longva et al., 2002; Figure 1, e and f).
To substantiate the differential stability of CD4-Ub chimeras, their plasma membrane turnover was determined next. Chimeras confined to the plasma membrane were labeled with anti-CD4 Ab on ice and then chased for 0–20 h at 37°C. Anti-CD4 Ab remaining at the cell surface was measured by the fluorescence generated in the presence of HRP-conjugated anti-mouse Ab and Amplex-Red. Cell surface remaining Ab was expressed as the percentage of the initial amount (Figure 2a and see Materials and Methods). The cell surface turnover of the monoubiquitinated CD4Tl-UbRG (T1/2 9.8 h) similar to CD4Tl (T1/2 16 h), was several-fold slower than that of the CD4Tl-Ub (T1/2 25 min) or CD4cc-URG (T1/2 34 min), representing poly- and multiubiquitinated cargo, respectively (Figure 2a).
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; Barriere et al., 2006; Hawryluk et al., 2006). These differences were also confirmed by measuring initial rates of anti-CD4 Ab uptake by the chimeras (Figure 2b). Endocytosis of both the multimeric CD4cc-UbRG and the polyubiquitinated CD4Tl-Ub was 3–4-fold faster (45–50%/5min) than CD4Tl, CD4Tl-UbRG, or CD4Tl-UbR (Figure 2b; Barriere et al., 2006). Importantly, the internalization of monoubiquitinated cargo (CD4Tl-UbRG and CD4Tl-UbR) was significant (13%/min and 8%/min) and comparable to that of the CD4Tl reporter molecule (12%/min) and thus conceivably was mediated by bulk flow endocytosis (Barriere et al., 2006). The relatively high cell surface density of monoubiquitinated cargo despite its slower internalization rate ensured sufficient Ab labeling of endocytic vesicles for immunolocalization and pH measurements (see below).
Poly-Ub and Multimeric-Ub Impedes Recycling and Targets Reporter Molecules into Lysosomes
Besides accelerated internalization, endosomal retention and lysosomal sorting may account for the rapid cell surface turnover of multi- and poly-ubiquitinated model cargoes as proposed for misfolded CFTR and yeast H+-ATPase (Sharma et al., 2004; Liu and Chang, 2006) and other ubiquitinated membrane proteins (Gruenberg and Stenmark, 2004; Traub and Lukacs, 2007). To address these possibilities, the recycling rates of chimeras were determined by the biotin-streptavidin sandwich technique as described in Materials and Methods. Although 34–38% of internalized CD4Tl and monoubiquitinated CD4Tl-UbRG returned to the cell surface in 5 min, the recycling efficiency of poly- and multiubiquitinated cargo was attenuated by two- to threefold (11–14%/5 min; Figure 2c). Intriguingly, preserving the terminal glycines in the CD4Tl-UbR decreased the recycling efficiency by 50% as compared with CD4Tl-UbRG (Figure 2, b and c), whereas it had no discernable effect on the internalization (Barriere et al., 2006), suggesting that terminal glycines may contribute to lysosomal sorting. These results, jointly, indicate that both accelerated internalization and defective recycling contribute to the rapid cell surface turnover of the poly-Ub– and multimeric-Ub–containing model proteins.
The destination of endocytosed chimeras was established first by colocalization studies using organellar markers and laser confocal fluorescence microscopy in HEK293 and COS-7 cells. Chimeras were labeled with anti-CD4 Ab and fluorophore-conjugated secondary Fab internalization and chased for 30 min in Ab-free medium at 37°C. CD4Tl-Ub and CD4cc-UbRG was predominantly targeted to lysosomes, identified by the fluid-phase marker dextran (Figure 3) or Lamp1 immunostaining (data not shown) and largely was excluded from recycling endosomes, visualized by FITC-Tf (Figure 3a). Conversely, endocytosed CD4Tl-UbRG carrying an unextendable Ub, similarly to CD4Tl, was sorted to recycling endosomes and excluded from lysosomes (Figure 3b). Considering that none of the above experiments allowed us to delineate the significance of ubiquitin configuration as lysosomal-sorting signal due its compounding effect on endocytosis (see Figure 2b), we designed an assay to measure the transfer kinetics of chimeras from early endosomes to lysosomes. The assay monitors the pHv and relies on the distinct pH of endolysosomal compartments.
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6.4–6.5) than sorting endosomes (pH 5.9–6.3), whereas maturation of early endosome into MVB/late endosome and lysosome is accompanied by their acidification to pH 5.5–6 and <5.5, respectively (Mukherjee et al., 1997; Supplementary Figure S1a). Therefore, cargo localization could be inferred from the pH of cargo-containing vesicles measured by fluorescence ratio image analysis (FRIA; see Materials and Methods), using cargo-bound FITC, a pH-sensitive fluorophore (Ohkuma and Poole, 1978).
To validate the assay, the destination of FITC-conjugated Tf, dextran, and EGF was established. The FRIA analysis confirmed that internalized Tf was confined to mildly acidic (pHv 6.35 ± 0.08, n = 3) recycling endosomes, whereas dextran and EGF were accumulated in acidic (pHv 4.60 ± 0.08 and 4.98 ± 0.15, n = 3) lysosomes (Figure 4a). These pHv values are similar to those reported for the respective organelles in fibroblast, BHK, and CHO cells (Mukherjee et al., 1997). The mean pHv value obtained in three or more independent experiments is indicated in the text and in Supplementary Table S1, whereas the result of a single experiment is depicted in figures.
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6.2) in 15 min and lysosomes (pH 4.8) during the subsequent 15-min chase (Figure 4b). CD4Tl accumulated slowly in recycling endosomes, consistent with its reduced internalization rate (Barriere et al., 2006) and was detected there even after 90-min chase, presumably due to multiple endocytic and exocytic rounds (Figure 4b and not shown). Similar results were obtained when the primary and secondary Abs were internalized for 1 h and chased for 30 min at 37°C (Figure 4c), a protocol used in subsequent studies.
Poly-Ub and Multimeric-Ub Are Recognized as Lysosomal Sorting Signals at Early Endosomes
To investigate Ub configuration(s) that may serve as an efficient lysosomal-targeting signal, the postendocytic trafficking of chimeras exposing poly-, multimeric-, or mono-Ub was compared with the labeling protocol described in Figure 4c. Although internalized CD4Tl-Ub was cleared from early endosomes and delivered to lysosomes during 30-min chase, CD4Tl-UbRG harboring a single Ub moiety was remained in a compartment with characteristic pHv of sorting endosomes (pHv 6.56 ± 0.08; Mukherjee et al., 1997) and was unable to reach the lysosome even after 90-min chase in both COS-7 and HEK293 cells (Figure 5a and Supplementary Table S1). CD4Tl-UbR also accumulated, predominantly, in early endosomes (pHv = 6.01 ± 0.05; Figure 5a and Supplementary Table S1). The limited lysosomal accumulation (10%) of CD4Tl-UbR indicated by the bimodal pHv distribution could be explained by the recruitment of the ubiquitination machinery via the terminal Gly residue that may contribute to lysosomal sorting (Hochstrasser, 2006). The modest delay of CD4Tl-UbG lysosomal delivery is consistent with this hypothesis (Figure 5a).
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; Figure 5b). Although IiT-UbRG accumulated at mildly acidic (pHv 6.57 ± 0.03) compartment, lysosomal delivery of IiT-Ub was completed during the 30-min chase (Figure 5b). Thus the inability of mono-Ub to be recognized as a lysosomal-sorting signal is independent of the reporter protein and was also confirmed with CD4 as cargo (see Supplementary Figure S4).
Remarkably, multimerization of CD4Tl-UbRG by two different means restored the chimera lysosomal delivery. Insertion of the cc tetramerization domain targeted CD4cc-UbRG and CD4cc-Ub similarly to CD4cc-UbR (data not shown) during the 30-min chase to highly acidic vesicles (pHv 4.94 ± 0.09, 4.82 ± 0.14, and 4.88 ± 0.16, respectively; Figure 5c and Supplementary Table S1). In contrast, CD4cc was confined to recycling endosomes (pHv 6.46 ± 0.09, n = 3), ruling out the role of the cc domain as a lysosomal-sorting motif (Figure 5c). As a second technique, cross-linking of CD4Tl-UbRG with primary Ab, biotinylated secondary Ab, and streptavidin was used to induce noncovalent clustering of the chimera. This method also rerouted the chimera, but not CD4Tl, from recycling endosomes into lysosomes (Figure 5d), implying that multimeric-Ub can be recognized as an efficient lysosomal-sorting signal.
Ub44Ile Is Critical for Lysosomal Sorting of CD4cc-UbRG
The hydrophobic patch around the Ub44Ile is required as the binding surface for Ub-binding domains, including the UIM of Hrs, STAM, epsin, and eps15 (Hicke, 2001; Fisher et al., 2003; Hicke and Dunn, 2003). To demonstrate the relevance of UIM-Ub interaction in the lysosomal targeting of the CD4cc-UbRG, Ub44Ile was mutated to Ala. According to pHv measurements, the CD4cc-UbRI44A was routed to recycling endosomes, in contrast to its wt counterpart, (pHv 6.38 ± 0.10, n = 3, Figure 5e). Concomitantly, the Ile44Ala mutation also restored the cell surface stability and recycling of the CD4cc-UbRI44A (Figure 2, a and c). These results confirm the inference that recognition of multiple Ub moieties is required for efficient lysosomal targeting and preventing constitutive recycling that leads to the metabolic destabilization of reporter molecules at the cell surface.
Inactivation of the E1 Enzyme Impedes the Lysosomal Delivery of the CD4Tl-Ub
To demonstrate that ubiquitin conjugation is indeed required for the lysosomal sorting of the CD4Tl-Ub, we took advantage of the ts20 cell line harboring a temperature-sensitive E1 Ub-activating enzyme. Complete down-regulation of the thermosensitive E1 enzyme could be achieved at 40°C in 3 h, verified by immunoblotting (Figure 6a). Ub conjugation was indispensable for the CD4Tl-Ub lysosomal targeting, because heat inactivation of the E1 enzyme dramatically impeded lysosomal accumulation of CD4Tl-Ub in ts20 cells (Figure 6b, top panel). In contrast, 40°C exposure had no effect on the lysosomal delivery in E36 cells containing the wild-type E1 enzyme (Figure 6b, top panels). Notably, efficient lysosomal targeting of the tetrameric CD4cc-UbRG was preserved at the nonpermissive temperature in ts20 cells, indicating that ubiquitination of the sorting machinery is not essential for ubiquitinated cargo sorting (Figure 6b, bottom panels).
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G. Lysosomal targeting of the CD4Tl-UbRG63K was partially restored during 30-min chase, because 58% of cargo reached the lysosomes (Figure 7a). Lysosomal delivery was less efficient for the 48K, reflected by 21% lysosomal accumulation of intracellular cargo and it was not detectable for the 6K or 29K substitutions (Figure 7a). Saturation of the endosomal sorting machinery cannot explain the recycling of UbRG6K, UbRG29K, and UbRG48K, because the cell surface density of the respective chimeras was increased by about fourfold, whereas their internalization was attenuated by 75% relative to CD4Tl-Ub (Supplementary Figure S1b). Thus K63 and K48 appear to serve as preferential acceptor sites for the first Ub conjugation.
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6.5, Figure 7c), conceivably by terminating chain extension. Neither wt nor UbR had discernable effects on Tf receptor recycling and Lamp1 or CD4cc-UbRG lysosomal sorting, ruling out nonspecific effects of Ub overexpression on lysosomal sorting (Supplementary Figure S2a). Although complete lysosomal accumulation of CD4Tl-Ub was achieved in the presence of UbR63K after 30-min chase, at least 90 min was required in case of UbR48K expression (Figure 7c). Considering the comparable expression level of Ub variants (Figure 7b and Supplementary Figure S2) and their conjugation to CD4Tl-Ub (Supplementary Figure S2), the simplest explanation is that the endosomal sorting machinery preferentially, but not exclusively, recognizes K63-linked Ub chain.
Preferential Binding of Hrs to K63-linked Polyubiquitin Chain
Hrs, a member of the ESCRT0, has been shown to play a pivotal role in ubiquitinated cargo recognition and lysosomal sorting at early endosomes (Hicke and Dunn, 2003; Raiborg et al., 2003). Inhibition of Hrs function delays lysosomal degradation of numerous ubiquitinated membrane proteins (Bache et al., 2003). Interaction of ubiquitinated CD4Tl-Ub with Hrs was demonstrated by coimmunoprecipitation. According to immunoblot analysis, the immunoprecipitates of CD4Tl-Ub, but not CD4Tl, contained Hrs (Figure 8a and data not shown). Furthermore, Hrs down-regulation by specific small interfering RNA (siRNA) inhibited the lysosomal delivery of CD4Tl-Ub, confirming Hrs involvement in the postendocytitc sorting of CD4Tl-Ub (data not shown).
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). Although Hrs was recovered with GST-2Ub, GST-3Ub, and GST-4Ub pulldown, minimal or no endogenous Hrs was associated with GST-Ub (Figure 8b). Eliminating one of the UIM recognition sites by Ile44Ala substitution in GST-Ub-Ub1A was sufficient to inhibit Hrs binding (Figure 8b, lanes 3 and 7). Increasing the distance between two Ub moieties by a flexible linker (GST-Ub-L-Ub) was permissive for Hrs binding and rendered the fusion protein accessible to the E1-activating enzyme (Figure 8b, lanes 3 and 6). Notably, the E1 Ub-activating enzyme was also bound to GST-Ub, implying that misfolding of the GST-Ub cannot account for the lack of its Hrs binding (Figure 8b, middle panel). In light of the dimerization propensity of the GST, these results support the notion that multivalent interactions are necessary for Hrs binding to ubiquitinated polypeptides. Homo- and hetero-oligomerization of Hrs with a subset of Ub-binding adaptors (e.g., STAM, eps15, and TSG101) may account for this phenomenon at early endosomes (Bache et al., 2003).
To assess the recombinant Hrs binding specificity, the association of mono-Ub, K63-, and K48-linked Ub chains with Hrs fused to maltose-binding protein (MBP) was determined in vitro. Comparable amounts of recombinant MBP-Hrs was bound to amylose beads and incubated with mono-Ub, K63-, and K48-linked Ub chains. Bound Ub was visualized by immunoblotting with anti-Ub Ab. Although neither the full-length nor a C-terminally truncated Hrs (Hrs298X) containing the UIM was able to bind detectable mono-Ub, a substantial amount of K63-linked poly-Ub chain was pulled down (Figure 8c). The recovery of K48-linked Ub chain was significantly attenuated (Figure 8c). Furthermore, disrupting Ub recognition by the S270A mutation in the UIM of Hrs (Miller et al., 2004), significantly diminished Ub binding (Figure 8c). These in vitro observations are consistent with preferential recognition of the K63-linked Ub chain by the Ub-dependent endosomal sorting machinery in vivo.
Lysosomal Sorting Signal of MARCH-IV Ubiquitinated CD4
Herpes viruses have been shown to encode viral homologues of mammalian proteins. Several candidates for mammalian homologues of modulator of immune recognition (MIR) family members share catalytic domains with viral E3 Ub ligases (Coscoy and Ganem, 2000; Coscoy et al., 2001). Based on their structural characters, MIRs have been designated as MARCH family members (Bartee et al., 2004). The E3 ligase activity of MARCH-IV ubiquitinates and down-regulates CD4 from the cell surface (Mansouri et al., 2003). To examine the relevance of Ub chain topology in MARCH-IV–induced CD4 down-regulation, three of four Lys residues was eliminated in the cytoplasmic tail of CD4 to prevent multiple Ub-chain formation (see Materials and Methods). In addition, we mutated the di-Leu motif to minimize Ub-independent sorting of CD4.
According to pHv measurements, CD4-1K was confined to recycling endosomes in the absence of MARCH-IV, whereas overexpression of MARCH-IV provoked the lysosomal delivery of CD4-1K during a 45-min chase (Figure 9a). Notably, the Lys-less CD4 (CD4-4R) variant that was inaccessible to ubiquitination remained at early endosomes even after 90-min chase (Figure 9A). Likewise, no lysosomal delivery of the CD4-1K was observed upon coexpression of MARCH-II that failed to down-regulate the CD4 (Bartee et al., 2004; data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Relevance of Polyubiquitination as a Lysosomal Targeting Signal
Although monoubiquitination was originally discovered as an endocytic signal of plasma membrane receptors and transporters (Ste2p, or galactose Gal2p and maltose transporters) in yeast (Lucero and Lagunas, 1997; Terrell et al., 1998; Horak and Wolf, 2001), recent analysis revealed that multiple Ub attachment can trigger more efficient cargo internalization than mono-Ub (e.g., Ste2p, the a-factor receptor Ste3p, the a-factor transporter Ste6p and the zinc transporter; Kolling and Hollenberg, 1994; Galan and Haguenauer-Tsapis, 1997; Terrell et al., 1998; Gitan and Eide, 2000; Roth and Davis, 2000). In animal cells poly- or multimeric-Ub, but not unextendable Ub modification was required to signal rapid endocytosis of model proteins (Barriere et al., 2006; Hawryluk et al., 2006). In accord, poly- or multiple-mono ubiquitination of numerous plasma membrane proteins was documented to be involved in the regulation of their cell surface density (for a review, see Traub and Lukacs, 2007). Ligand-induced down-regulation of TrkA receptor, as well as the MIR K3-mediated MHC I ubiquitination requires K63-linked poly-Ub chain formation (Duncan et al., 2006; Geetha et al., 2005). On the other hand, the activated dimeric tyrosine kinase receptor (e.g., EGFR) undergoes both multiple mono- and poly-ubiquitination (Haglund et al., 2003; Geetha et al., 2005). Polyubiquitination of MHC II complex is also critical in limiting antigenic peptide presentation (Ohmura-Hoshino et al., 2006; Shin et al., 2006; van Niel et al., 2006). Virally encoded E3-Ub ligases of the MIR family polyubiquitinates numerous immune recognition molecules, including B7.2, ICAM-I, CD1d, CD4, and interferon 
receptor 1 (Lehner et al., 2005; Li et al., 2007). Polyubiquitination of channels, transporters, and other receptors (AQP2, ENac, 2 adrenergic receptor-arrestin complex, TRPV4, CFTR, and ClC-5) is also indispensable for internalization (Staub et al., 1997; Shenoy et al., 2001; Hryciw et al., 2004; Sharma et al., 2004; Kamsteeg et al., 2006; Wegierski et al., 2006; Wiemuth et al., 2007). These observations, collectively, highlight the prevalence and functional significance of poly- and multiple-mono ubiquitination as autonomous endocytic signal and reinforce their relevance as possible lysosomal-targeting signal.
Monitoring Polyubiquitination or Multiple-mono Ubiquitination Requirement for Lysosomal Cargo Sorting by Vesicular pH Determination
Considering that cargo ubiquitination signals both internalization and lysosomal targeting from early endosomes and accelerated internalization per se could lead to enhanced endolysosomal cargo flux (Bonifacino and Traub, 2003), it was imperative to design an assay that monitors the dynamics of endocytosed cargo destination. This was achieved by implementing the FRIA assay to determine the postendocytic localization of cargo molecules based on the characteristic acidification pattern of endolysosomal compartments (Mukherjee et al., 1997). Although sorting and recycling endosomes, as well as MVB and lysosomes have distinct luminal pH, the TGN pH 6.2–6.6 overlaps with that of recycling endosomes (Sun-Wada et al., 2004). Therefore, the recycling propensity of cargoes was also monitored by coimmunolocalization with the Tf receptor (TfR; Figure 3) and quantification of recycling efficiency by the biotin-avidin sandwich technique (Figure 2c).
Several lines of evidence support our conclusion that internalized model proteins and the CD4 require poly- or multimeric Ub modification for efficient MVB/lysosome targeting from endosomes. 1) Preventing Ub-chain conjugation by fusing unextendable Ub (UbRG) to the truncated CD4 or Ii, similar to overexpression of UbR with CD4Tl-Ub or CD4, was sufficient to reroute these cargoes to recycling endosomes, according to morphological, biochemical, and functional assays. On the other hand, constitutive recycling of the CD4Tl-UbRG could be converted into lysosomal targeting by introduction a single Lys residue (K63) into UbRG. The recycling propensity of CD4 chimeras was inversely proportional to their lysosomal-targeting efficiency, substantiating the role of endosomes in defining the destination of ubiquitinated cargo at sorting endosomes. 2) Thermodenaturation of the E1 Ub-activating enzyme severely inhibited the lysosomal delivery of the polyubiquitinated CD4Tl-Ub in ts20 cells. This inhibition was specific, because E1 inactivation failed to interfere with the recycling of TfR and the lysosomal delivery of Lamp1 or CD4cc-UbRG, cargo molecules with ubiquitination-independent sorting signals. 3) Multimerization of monoubiquitinated CD4Tl-UbRG, accomplished by genetic and biochemical means, restored the lysosomal targeting of chimera. This implies that the Ub-dependent endosomal sorting machinery has the plasticity to recognize both Ub chain and multimeric-Ub. The latter phenomenon has particular significance in the desensitization of activated dimeric tyrosine kinase receptors that undergo multiple mono- and polyubiquitination (Haglund et al., 2003; Geetha et al., 2005) and oligomeric polypeptides with monoubiquitinated subunits such as the ROMK1 K+ channel (Lin et al., 2005). Notably, constitutive trimerization failed to redirect the Ii-UbRG to the lysosome, whereas tetrameric mono-Ub was rapidly delivered into MVB/lysosomes in the context of CD4cc-UbRG. This suggests that exposure of at least four Ub moieties is required for lysosomal sorting, although we cannot rule out the possibility that trimerization masked the UIM binding surface in the Ii-UbRG. 4) Finally, we showed that disrupting the Ub-UIM–interacting surface by the UbI44A mutation prevented the CD4Tcc-UbRG MVB/lysosomal sorting and restored recycling, corroborating the notion that Ub-binding proteins are critical in the lysosomal sorting of chimeras.
Recognition of Poly- and Multiubiquitinated Cargo in Early Endosomes
The observation that the Ub-dependent endosomal sorting machinery preferentially recognizes poly- and multiubiquitinated over monoubiquitinated cargo is not completely surprising in light of biochemical and functional characteristics of Ub recognition as an endocytic signal (Williams and Urbe, 2007). Although decoding Ub as a sorting motif relies on different sets of adaptors at various cellular locations, the primary recognition of the Ub Ile44 hydrophobic patch is mediated by a common 
-helical Ub-binding domain, the UIM of adaptors both at the cell surface and at sorting endosome (Hicke et al., 2005; Hurley and Emr, 2006). Eps15/15R and epsin, plasma membrane clathrin adaptors, contain two and three UIMs, respectively (Bonifacino and Traub, 2003; Hicke and Dunn, 2003). The endosomal adaptor STAM has a single UIM, whereas Hrs contains the double-sided variant of UIM, the DUIM, which is capable of binding two Ubs simultaneously with affinities comparable to that of the UIM (Urbe et al., 2003; Hirano et al., 2006). In vitro–binding studies revealed that the UIMs bind to mono-Ub with a surprisingly low affinity in the 0.1–2 mM range (Shekhtman and Cowburn, 2002; Fisher et al., 2003). This may explain the inability of mono-Ub to serve as internalization (Barriere et al., 2006) and lysosomal-sorting signal for transmembrane cargo in mammalian cells (Duncan et al., 2006). Synergistic mechanisms are likely involved to enhance the low-affinity UIM-Ub interaction in vivo. These may include multimerization of adaptor molecules, multiplication of Ub-binding domains in adaptors, concentration of adaptor/cargo at specific sorting platforms, and modulation of UIMs affinity (Cupers et al., 1997; Chen et al., 1998; Raiborg et al., 2002; Sachse et al., 2002; Bache et al., 2003; Sugiyama et al., 2005; Hurley and Emr, 2006).
In respect of Ub recognition as a lysosomal-sorting signal, stable complex formation between Hrs and STAM (also termed ESCRT0) can facilitate the binding of multiple Ub moieties. The yeast orthologues of Hrs and STAM–Vps27 and Hse1–contain two tandem and a single UIM, respectively (Hurley and Emr, 2006; Williams and Urbe, 2007). The Vps27/Hse1 complex associates with multiple Ubs, situated at different distances from the membrane according to Monte Carlo simulation and can undergo large conformational changes to accommodate multiple Ub moieties (Prag et al., 2007). Considering that the VHS domain of STAM can also recognize Ub (Mizuno et al., 2003), heterodimerization of Hrs/STAM would expose four Ub-binding sites. Furthermore, the oligomerization tendency, indicated by the hexamerization of recombinant Hrs (Pullan et al., 2006) and isolation of endogenous Hrs in 500–600-kDa complexes from HeLa cell lysate (unpublished observation), may further enhance the number of functional Ub-binding sites in ESCRT0. Finally, concentration of both ubiquitinated cargo and the Hrs/STAM complex at the endosomal double-layered coat by clathrin recruitment and PtdIn3P binding of the Hrs FYVE domain could increase the association of the ubiquitinated cargo and the ESCRT0 (Sachse et al., 2002). Remarkably, homo- or hetero-oligomerization of eps15/15R/epsin adaptors may also serve to enhance their avidity to poly-Ub chain (Cupers et al., 1997; Chen et al., 1998; Polo et al., 2002; Miller et al., 2004; Sugiyama et al., 2005; Hawryluk et al., 2006).
We showed that the K48-linked poly-Ub chain is poorly recognized by recombinant Hrs in vitro, despite the 2–3-fold slower lysosomal-targeting kinetics of the CD4Tl-Ub in the presence of overexpressed UbR48K (Figures 7d and 9b, and Supplementary Figure S4). These observations are at variance with the negligible effect of UbR48K and UbR63K overexpression in the internalization of the CD4Tl-Ub (Figure 7e), but in line with the modest Ub-chain selectivity of the epsin UIMs (Wang and Struhl, 2005; Hawryluk et al., 2006). The relatively limited Ub-chain specificity of the Ub-dependent endosomal-sorting machinery compared with recombinant Hrs could be explained by a combination of factors. The substrate specificity of Hrs could be altered by association with STAM, clathrin heavy chain, and PtdIns3P (Williams and Urbe, 2007). Furthermore, posttranslational modification, such as monoubiquitination and phosphorylation of Hrs/STAM, may play a role in the ESCRT0 binding specificity in vivo (Row et al., 2005; Hoeller et al., 2006). Finally, decreased availability of STAM due to K48 poly-Ub chain conjugation and proteolysis via proteasomes may influence lysosomal-sorting kinetics of ubiquitinated cargo (Williams and Urbe, 2007).
Modulation of Lysosomal Sorting Fidelity by the Extent of Poly- and Multiubiquitination of Cargo Molecules
We envision two advantages of poly- and multimeric-Ub as a lysosomal-sorting signal over mono-Ub. First, the redundant nature of ubiquitin modification may permit flexibility for substrates and E2/E3 enzyme pairing, as well as for the Ub chain configuration. Although some plasma membrane proteins undergo K63-linked Ub chain attachments, both K63- and K48-linked Ub chains were detected in activated EGFR (Huang et al., 2006). Preferential K48-linked over K63-linked Ub chain conjugation would delay the desensitization of activated receptors, based on the significantly slower lysosomal transfer kinetics of cargoes with K48-lined Ub chain. Second, attachment of Ub chain or multimeric-Ub may allow fine-tuning of the sorting efficiency by regulating signal recognition via the opposing activity of Ub ligase(s) and deubiquitinating enzyme(s) (DUB) at endosomes (Amerik and Hochstrasser, 2004) as opposed to the bimodal regulation with a single Ub-sorting motif. This modulation would be conceptually similar to the role that ligases and deubiquitinating enzymes play in influencing the destruction of polyubiquitinated polypeptides by the proteasome (Crosas et al., 2006).
Association of DUB and E3 Ub ligase with constituents of the ESCRT0 have been documented in both yeast and mammalian cells (Clague and Urbe, 2006). In yeast, the Vps27/Hse1 complex binds to the DUB Ubp7, as well as the Rsp5 ligase to control sorting efficiency into vacuoles (Ren et al., 2007). In mammalian cells, the SH3 domain of STAM recruits UBPY (ubiquitin isopeptidase Y; Kato et al., 2000) and AMSH (associated molecule with SH3 domain of STAM; McCullough et al., 2006). The isopeptidase activity of UBPY cleaves both the K63- and K48-linked Ub chains, whereas AMSH shows preferential activity toward the K63-linked Ub chain. Thus AMSH serves as an off switch, whereas UBPY, depending on configuration of the primary Ub chain and on the E3 ligase activity in the sorting complex, may accelerate or delay lysosomal cargo delivery.
Although our FRIA experiments allowed us to follow the endosomal sorting of internalized ubiquitinated cargo molecules and highlighted the necessary and sufficient Ub configuration for lysosomal targeting, the cellular location and the molecular determinants of cargo-specific ubiquitination remains to be established. Nevertheless, the FRIA technique complements existing methodologies to follow the destination of endocytosed plasma membrane proteins at relatively high temporal and spatial resolution and will help to elucidate the molecular basis for the recognition of sorting signals at endosomes.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: G. L. Lukacs (glukacs{at}sickkids.ca)
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3 independent experiments are summarized in Supplementary Table S1. (b) The kinetic of CD4Tl-Ub lysosomal delivery. Anti-CD4 antibody and FITC-conjugated secondary Fab was bound to transfected HEK293 cells for 1 h at 0°C. Then the temperature was raised to 37°C for 1, 15, or 30 min, and the pHv was measured by FRIA. (c) CD4Tl- and CD4Tl-Ub–expressing HEK293 cells were allowed internalize anti-CD4 primary Ab and FITC-conjugated secondary Fab for 1 h at 37°C and were chased for 30 min before FRIA. Similar results were obtained in COS-7 cells (data not shown).
