Cullin-3 Regulates Late Endosome Maturation

Cullin-3 regulates late endosome maturation

Jatta Huotaria,1, Nathalie Meyer-Schallera,1,2, Michaela Hubnera, Sarah Stauffera, Nadja Kathedera,3, Peter Horvathb, Roberta Mancinia, Ari Heleniusa,1,4, and Matthias Petera,1,4

aInstitute of Biochemistry and bLight Microscopy Center, Department of Biology, ETH Zurich, CH-8093 Zurich, Switzerland

Contributed by Ari Helenius, December 3, 2011 (sent for review July 29, 2011)

Cullin-3 (Cul3) functions as a scaffolding protein in the Bric-a-brac, numerous effector proteins from the cytosol (19, 20). There is Tramtrack, Broad-complex (BTB)–Cul3–Rbx1 ubiquitin E3 ligase com- also a further reduction in luminal pH and formation of intra- plex. Here, we report a previously undescribed role for Cul3 com- luminal vesicles (ILVs). For many cargo proteins, such as EGFR, plexes in late endosome (LE) maturation. RNAi-mediated depletion inclusion within ILVs depends on ubiquitination of the cytosolic of Cul3 results in a trafficking defect of two cargoes of the endoly- domain and recognition by components of the endosomal sorting sosomal pathway, influenza A virus (IAV) and epidermal growth complex required for transport (ESCRT) machinery (21, 22). factor receptor (EGFR). IAV is able to reach an acidic endosomal com- Here, we demonstrate that Cul3 depletion resulted in non- partment, coinciding with LE/lysosome (LY) markers. However, it productive accumulation of two endocytic cargoes, IAV and EGF/ remains trapped or the capsid is unable to uncoat after penetration EGFR in LEs/LYs, accompanied by the accumulation of LEs with into the cytosol. Similarly, activation and subsequent ubiquitination abnormal morphology. Together these results suggest a unique of EGFR appear normal, whereas downstream EGFR degradation is role for Cul3 as a critical regulator of LE maturation. delayed and its ligand EGF accumulates in LE/LYs. Indeed, Cul3-de- Results pleted cells display severe morphological defects in LEs that could account for these trafficking defects; they accumulate acidic LE/LYs, Cul3 Depletion Causes a Defect in IAV Entry. To identify host cell and some cells become highly vacuolated, with enlarged Rab7-pos- factors of IAV, our collaboration partner, 3-V Biosciences, Inc., itive endosomes. Together, these results suggest a crucial role of Cul3 performed an siRNA screen in A549 cells and found Cul3 as in regulating late steps in the endolysosomal trafficking pathway. a possible regulator of infection. We validated these results with two siRNAs against Cul3 that efficiently decreased Cul3 expression (Fig. 1 A and B). As measured by viral nuclear protein membrane trafficking | multivesicular body | virus entry | endocytosis | CELL BIOLOGY fl (NP) expression, IAV infection in Cul3-depleted A549 cells was in uenza virus decreased up to 93%, compared with control. Vesicular stomatitis virus (VSV), which unlike IAV fuses in EEs (23, 24), was not y serving as a scaffolding subunit in a large family of E3 ubiq- affected. Depletion of another cullin family member, Cul1, did Buitin ligases, cullins play a central role in the regulation of not affect IAV infection, although it did elevate VSV infection a multitude of cellular functions (1). The cullin family member, (Fig. 1 A and B). These results indicated that IAV, a virus that Cul3, is responsible for joining the ligase activity-bearing subunit penetrates from acidic LEs, depends on Cul3 for infection. Rbx1 with a BTB-containing protein. BTB proteins serve as As shown in Fig. 1C, we found that the virus was endocytosed in adaptors for the recognition of a wide variety of specific substrate Cul3-depleted cells and entered cytoplasmic vacuoles where HA proteins in processes such as cell cycle, apoptosis, antioxidative underwent the acid-induced conformational change, detected with responses, and cytoskeleton dynamics (2–5). the conformation-specific antibody, A1 (25). No acidified HA was Recently, we found that Cul3 was present in detergent-resistant observed in cells depleted of a subunit of the vacuolar-type H+- membrane fractions (DRMs), by the action of lipid-modified ATPase (V-ATPase), ATP6V1B2. After penetration into the cy- DCNL3, a protein that promotes Cul3 activity through neddyla- tosol, uncoating of the viral capsid can be detected by the diffuse tion (6). Moreover, two BTB proteins, Rabankyrin-5 and staining of M1 in the cytoplasm (15). In contrast to control cells, RhoBTB3, are known regulators of membrane trafficking (7, 8). Cul3-depleted cells showed an endosome-like distribution of M1 These observations suggested that Cul3-mediated ubiquitination staining (Fig. 1D). Likewise, NP accumulated in endosome-like might have undetected membrane-associated functions. To in- structures in the cytoplasm, and not in the nucleus as in control vestigate this possibility, we monitored the internalization and cells (Fig. 1E) (15). Except for the conversion of HA to the acid intracellular fate of two endocytosed cargo in Cul3-depleted cells: conformation, the overall picture resembled that of V-ATPase the EGF/epidermal growth factor receptor (EGFR) complex and inactivation. We found most of the virus to reach LEs, identified by influenza A virus (IAV). the colocalization of acidified A1-positive virus particles with EGFR belongs to a cohort of plasma membrane proteins tar- vesicles containing the LE/LY marker LAMP1 and the LE/TGN geted to early endosomes (EE) from where a fraction is brought marker cation-independent mannose-6-phosphate receptor (CI- via late endosomes (LEs) to lysosomes (LYs) for degradation (9). M6PR). In contrast, virus particles were not found in vesicles During infectious entry in host cells, IAV particles follow a similar bearing the EE marker EEA1 (Fig. 2A). Similarly, after 3 h when pathway except that when the virus reaches the LEs, the low pH (pH 5.4–4.9) induces an irreversible conformational change in – the membrane fusion protein, the hemagglutinin (HA) (10 13). Author contributions: J.H., N.M.-S., A.H., and M.P. designed research; J.H., N.M.-S., M.H., This conformational change triggers the fusion of the viral enve- S.S., N.K., and R.M. performed research; P.H. contributed new reagents/analytic tools; J.H., lope with the limiting membrane of the endosome. The viral N.M.-S., M.H., S.S., N.K., P.H., and R.M. analyzed data; and J.H., N.M.-S., A.H., and M.P. ribonucleoproteins (vRNPs) and the matrix protein (M1) are re- wrote the paper. leased into the cytosol where they dissociate from each other. This The authors declare no conflict of interest. uncoating allows the vRNPs to be imported into the nucleus 1J.H., N.M.-S., A.H., and M.P. contributed equally to this work. – where replication takes place (14 16). 2Present address: Department of Biomedicine, University of Basel, CH-4058 Basel, Cargo sorting from EEs into LEs and delivery into the LY Switzerland. fi compartment requires a number of membrane ssion and fusion 3Present address: Department of Biochemistry, Institute for Cancer Research, The Norwe- events, as well as the maturation of LEs preparing them for in- gian Radium Hospital, N-0310 Oslo, Norway. teraction with LYs (17, 18). Maturation occurs through a variety 4To whom correspondence may be addressed: E-mail: [email protected] or of protein- and lipid-based remodeling events including a switch [email protected]. from Rab5 to Rab7, a PIKfyve-dependent phosphatidylinositide This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. conversion from PtdIns(3)P to PtdIns(3,5)P2, and recruitment of 1073/pnas.1118744109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1118744109 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 siControl siCul3 A Infection B A 2.5 IAV VSV 1.83 kDa siControlsiCul3-1siCul3-2 2.0 75 Cul3 A1 colocalization 50 EEA1 1.00 1.28 EEA1 LAMP1 CI-M6PR 1.5 1.02 Actin 1.00 1.05 0.8 1.0 0.22 Infected cells 0.5 0.07 0.6 kDa siControlsiCul1 0.0 Cul1 A1 75 0.4 50 LAMP1 siCul1 α-Tubulin siControl siCul3-1 siCul3-2 0.2

0.0 C fraction of colocalized A1 siControl siCul3 Acidification (2h) CI-M6PR

siControl 1.03 1.2 1.00 1.0 siCul3 0.8 B 0.6 DRAQ5 0.4 M1 colocalization A1 signal 0.01

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1.00 CI-M6PR 1.0 0.8 0.6 DRAQ5 Endocytosis bypass 0.4 0.10 0.02 C 0.2 M1 pH 4.5 pH 6.8 infection 0.0 1.2

Dispersed M1 phenotype 1.0 siCul3 siControl 0.8 ATP6V1B2 si 0.6

siATP6V1B2 siCul3 0.4 infected cells 0.2 E 0.0 siControl siCul3 siATP6V1B2 Nuclear import (5h)

siControl Fig. 2. IAV is blocked in LAMP1-positive LEs/LYs in Cul3-depleted cells. (A 1.2 1.00 and B) Colocalization of IAV with endosomal markers. siRNA-transfected 1.0 0.8 A549 cells were infected for 1 (A)or3(B) h with IAV, and stained with A1 0.6 0.25 (A)orM1(red)(B) and either EEA1, CI-M6PR or LAMP1 (green) anti-

DRAQ5 0.4 0.02 μ ﬁ 0.2 bodies. Shown are single confocal sections. (Scale bars: 2 m.) Quanti - NP

NP positive nuclei 0.0 cation of colocalization shows the mean fraction of A1 or M1-positive virus particles that colocalized with the endosomal markers EEA1, CI- siCul3 siControl M6PR, or LAMP1 with SEM from at least three independent experiments. ATP6V1B2 si (C) Endocytosis bypass assay. Shown is the mean infection for corre-

siATP6V1B2 siCul3 sponding type of experiment; acid bypass (filled bars) or normal infection (open bars), normalized to siControl with SEM from three independent fi Fig. 1. Depletion of Cul3 allows delivery of IAV to acidi ed compartments experiments. but blocks IAV uncoating. (A) Virus infection in A549 cells measured by IAV NP and VSV-G protein production. Shown is mean infection, normalized to control siRNA with SEM from three independent experiments. (B) Verification of at the plasma membrane (27, 28), which allows vRNP delivery fi fi knockdown ef ciency in A549 cells. (C) IAV HA acidi cation at 2 h after in- directly into the cytoplasm without endocytosis and vesicular fection (p.i.), as measured by staining with A1 antibody. Nuclei were stained trafficking. When the bypass was performed in Cul3-depleted cells, with DNA dye, DRAQ5. Quantification shows A1 positive area per cell, normalized to siControl. (D) Uncoating assay of IAV, with anti-M1 staining, 3 h p.i. infection was rescued to a level comparable to cells treated with Quantification shows cell classification, based on M1 dispersal phenotype, control siRNAs or siATP6V1B2 (Fig. 2C). We concluded that fi Cul3-depletion caused specific defects in the cellular inter- classi ed as uncoated, and normalized to siControl. (E) Nuclear import assay fi of IAV NP, 5 h p.i. Quantification shows percentage of NP positive nuclei, nalization and traf cking machinery, blocking virus infection at a normalized to siControl. For C–E, single confocal sections from the midpoint of prepenetration step. cells are shown. Bars for all graphs represent means ± SEM from at least three The results suggested that Cul3 is not required for viral traf- independent experiments. siRNAs against a subunit of V-ATPase (ATP6V1B2) ficking to LEs or for the acidification of HA in late endocytic were used as a negative control for all three assays. (Scale bars: 10 μm.) compartments. However, it is needed for the productive penetration of the vRNPs and M1 from these compartments or for uncoating of the viral capsids in the cytoplasm. most viruses had escaped from endosomes in control cells (Fig. 1D), the M1 in Cul3-depleted cells was still associated with Cul3 Depletion Leads to Accumulation of EGF and EGFR. To test LAMP1 and CI-M6PR-, but not EEA1-positive, vesicles (Fig. 2B). whether Cul3 depletion affected endocytosis and processing of Because CI-M6PR is absent from lysosomes (26), we conclude that physiological cargo destined to LEs and lysosomal degradation, we virus particles were trapped in LEs upon Cul3 depletion. analyzed the fate of EGFR and its ligand EGF in HeLa cells. Finally, we tested whether the block in IAV infection upon Cul3 Fluorescence microscopy showed that the amount of EGF-Tex- depletion could be bypassed by low pH-induced viral penetration asRed (EGF-TxRed) accumulating in cytoplasmic vesicles almost

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1118744109 Huotari et al. Downloaded by guest on September 23, 2021 doubled upon Cul3 depletion (Fig. 3A). The difference was most Cul3-depleted cells, in contrast to c-Cbl depletion (Fig. 4D and pronounced 30–60 min after internalization (Fig. 3A, Right). Fig. S2A). Whereas ubiquitination appeared similar to control We also observed an increase in the amount of the receptor, conditions, deubiquitination of EGFR may have been slightly EGFR, in cells transfected with siCul3 (Fig. 3B and Fig. S1A). An delayed in the absence of Cul3. However, although Cul3 has re- increase in protein level after Cul3 depletion was also observed for cently been proposed to regulate several deubiquitinating enzymes another receptor-tyrosine kinase, insulin-like growth factor re- (DUBs) (29), we found that levels of c-Cbl and the DUBs, AMSH ceptor (IGF1R), whereas no increase was detected for transferrin and UBPY, known to be involved in lysosomal degradation of receptor (TfR), which unlike EGFR and IGF1R is not destined for EGFR (30), were unaffected by Cul3 depletion (Fig. S2 B and C). degradation in LYs (Fig. S1B). In addition, overexpression of Cul3 We conclude that Cul3 is required for efficient EGFR deg- did not affect the level of EGFR (Fig. 3C), suggesting that Cul3 is fi fi radation, but does not regulate the activation and ubiquitination required but not limiting for ef cient EGF and EGFR traf cking fi and degradation. of EGFR after ligand stimulation. These ndings are consistent

Cul3 Regulates Lysosomal Degradation of EGFR, but Not Its Activation and Ubiquitination. The ninefold increase in EGFR protein levels A EGFR mRNA (Fig. 3B) was accompanied by a mere twofold elevation in 2.5 1.9 mRNA levels (Fig. 4A), suggesting that the receptor may not be 2 1.5 efﬁciently down-regulated by degradation, i.e., that there could 1 be a problem for example with trafﬁcking to LYs. To directly 1 0.5

assess degradation of EGFR, we used cycloheximide (CHX) to relative to siControl 0 block protein translation (Fig. 4B). Whereas in control cells the siControl siCul3 relative amount of EGFR was decreased 2 h and 4 h after CHX addition, EGFR degradation was signiﬁcantly delayed in Cul3- B EGFR protein siControl siCul3 depleted cells. Together these results suggested that accumula- siControl siCul3 CHX 024 024 treatment (h) 1.2 ** kDa tion of EGFR and its ligand EGF in Cul3-depleted cells resulted 1 EGFR from delayed degradation of endocytic cargo destined to 150 0.8 0.6 the LYs. 75 Cul3 0.4 For EGFR to be sorted into the degradative pathway, it needs 0.2 35 GAPDH 0 CELL BIOLOGY to be activated by its ligand EGF, which induces receptor relative to 0h siRNA 0 h 2 h 4 h dimerization, autophosphorylation, and ubiquitination (9, 21). Time of CHX EGFR was phosphorylated after EGF addition with similar ki- treatment (h) netics in Cul3-depleted cells as in control cells (Fig. 4C). We also C siControl siCul3 observed EGFR to be ubiquitinated upon EGF stimulation in kDa 0 5 30 90 180 240 360 0 5 30 90 180 240 360 EGF (min)

150 EGFR

p-EGFR A siControl siCul3 150 EGF WGA 75 Cul3 5 EGF signal intensity

5 min siControl 50 α-Tubulin 4 siCul3 3 D siControl siCul3 2 kDa 0 5 30 90 180 0 5 30 90 180 EGF (min) arbitrary units 30 min 1 Immunoprecipitation

0 250 5 1530456090180 Time after EGF addition (min) Ubiquitin

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B C Input kDa siControlsiCul3 kDa HA HA-Cul3 150 EGFR

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75 Cul3 75 * Cul3 75 HA (Cul3)

50 α-Tubulin 50 α-Tubulin Fig. 4. EGFR lysosomal degradation is delayed, whereas phosphorylation and ubiquitination occur upon Cul3 depletion. (A) EGFR mRNA levels from * Nedd8-Cul3 Cul3 siRNA transfected HeLa cells, quantified by quantitative RT-PCR, normalized to GAPDH, relative to siControl with corresponding SEM from at least three Fig. 3. Labeled EGF and EGFR accumulate in Cul3-depleted cells. (A) EGF independent experiments. (B) Degradation of EGFR. siRNA-transfected HeLa intracellular accumulation over time. siRNA-transfected HeLa cells were cells were untreated (0 h) or treated with 10 μM CHX for 2 or 4 h and ana- starved overnight and treated with 100 ng/mL EGF-TxRed for 5 min (pulse) lyzed for EGFR protein levels. Graph shows mean EGFR protein amounts, followed by replacement to full medium (chase). The minutes on images normalized to GAPDH, relative to 0 h of corresponding siRNA with SEM from and on the x axis of graph represent total time from addition of EGF-TxRed five independent experiments. **P < 0.01. (C) Activation of EGFR. siRNA- (5 min pulse plus chase). Quantification shows mean EGF-TxRed signal per transfected HeLa cells were serum starved, stimulated with 100 ng/mL EGF, cell, normalized to siControl 5 min with SEM from at least three independent and analyzed for EGFR and p-EGFR (EGFR-pY1173) levels. (D) Ubiquitination experiments. (Scale bars: 10 μm.) Immunoblotting for EGFR of HeLa cells of EGFR. Cell lysates (input), treated as in C, were immunoprecipitated with transfected with (B) siRNAs (star in Cul3 blot marks neddylated form of Cul3) EGFR antibodies (under limiting conditions to pull down equal amounts of or (C) HA-Cul3 or HA-control plasmid. EGFR) and analyzed for EGFR ubiquitination by immunoblotting.

Huotari et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 with a role of Cul3 in endosome maturation or trafficking, as A 30min EGF described above for IAV. EGF EEA1 EGF Accumulates in LE/LYs upon Cul3 Depletion. To better define the EGF colocalization with EEA1 0.06 possible defect in EGF/EGFR endosomal trafficking in Cul3- siControl depleted cells, we assayed the kinetics and localization of EGF- 0.05 siCul3

ﬂ siControl TxRed by immuno uorescence with different endosomal mark- 0.04 ers. Trafﬁcking of EGF-TxRed out of EEA1-positive EEs was mostly unaffected (Fig. 5A), except for a slight delay at 30 min 0.03 after uptake. Moreover, we did not detect a major difference in 0.02

the kinetics of EGF-TxRed colocalization with HRS, a compo- 0.01 nent of the ESCRT-0 complex present on EEs (Fig. 5B), implying fi fi 0 that Cul3 depletion does not signi cantly affect traf cking of EEA1 with colocalized of EGF Fraction 5 10152030456090180 EGF at the level of EEs. The general increase of EGF-TxRed siCul3 Time after EGFaddition (min) colocalization with EEA1 and HRS likely reflected increased amounts of internalized EGF-TxRed (Fig. 3A). In contrast, EGF- TxRed colocalization with LAMP1-positive LE/LY compartments was significantly increased in Cul3-depleted cells 60 and 90 B 30min EGF min after internalization compared with maximum at 30 min (Fig. EGF HRS 5C and Fig. S3A), consistent with the delay in EGF/EGFR deg- EGF colocalization with HRS radation. Finally, it is unlikely that Cul3 regulated transport of 0.05 acid hydrolases from the TGN to endosomes as cleavage of the siControl cathepsin D precursor to its active form was unaffected in Cul3- 0.04 siCul3 siControl depleted cells (Fig. S3B). Cathepsin D cleavage requires trans- 0.03 port of the enzyme to LE/LYs and acidification (31). These results suggested that degradation of EGFR/EGF 0.02

complexes was delayed. Like IAV, they were trapped in LE/LYs. 0.01

Cul3 Depletion Distorts LE Morphology. The late endocytic organelles 0

Fraction of EGF colocalized with HRS with colocalized EGF of Fraction 5 10152030456090180 were clearly expanded with respecttosizeandnumberinCul3-de- Time after EGF addition (min) pleted HeLa and A549 cells, as visualized by staining with the LE/LY siCul3 markers LAMP1, CD63, and the lipid lyso(bis)phosphatidic acid (LBPA)/ bis(monoacylglyceryl)phosphate (BMP) (Fig. 6A and Fig. S4A). A comparable expansion of LE/LY compartments was de- 60min EGF tected when cells were depleted of known regulators of ILV for- C EGF mation and endosome maturation such as the ESCRT-0 component LAMP1 HRS, Rab7, and the ATP6V1B2 subunit of V-ATPase (Fig. S4B), EGF colocalization with LAMP1 suggesting that Cul3 regulates an aspect of endosome maturation. 0.06 siControl ** siCul3 Using live cell imaging, we found that some of the Cul3-de- 0.05 *

pleted cells contained numerous large, spherical vacuoles (Fig. siControl 0.04 6B). The vacuoles stained positive with LysoTracker, a marker for acidic compartments, although the largest showed somewhat 0.03 weaker staining. Consistent with the elevated LE/LY marker 0.02

staining (Fig. 6A and Fig. S4A), we observed a general increase 0.01 in LysoTracker-positive vacuoles (Fig. 6B). fi 0 The enlarged vacuoles probably represented modi ed LEs of EGFFraction colocalized with LAMP1 5 10152030456090180 because they were Rab7 positive and largely devoid of Rab5 (Fig. siCul3 Time after EGF addition (min) 6C). In stably transfected HeLa cells expressing EGFP-Rab7, ≈30% acquired enlarged Rab7-positive vacuoles upon Cul3 depletion, compared with 6% in siControl cells (Fig. 6D). Moreover, the fluid-phase marker, Dextran-Alexa Fluor 488 accumulated in Fig. 5. EGF-TxRed colocalization with LAMP1-positive LEs/LYs is prolonged the enlarged endosomes, indicating that the vacuoles were con- upon Cul3 depletion. (A–C) siRNA-transfected HeLa cells were serum starved, pulsed for 5 min with EGF-TxRed (100 ng/mL), and chased with full medium. nected to the endocytic system (Fig. 6E). fi Two pharmacological inhibitors were found to induce a similar Cells were xed and stained with antibodies against EEA1 (A), HRS (B), or LAMP1 (C). n > 200 cells for each timepoint and condition. Bars represent vacuolation effect (Fig. S4C). MLN4924 inhibits the Nedd8-ac- means with SEM from at least three independent experiments. P values were tivating enzyme, required to activate cullin-based ligases (32). fi – calculated between siControl and siCul3 colocalization and compared with The percentage of cells lled with enlarged EGFP-Rab7 positive maximum values (Fig. S3A). The P values from EGF colocalization with EEA1 endosomes increased over time, reaching 80% after 24 h of drug and HRS, or other timepoints of LAMP1 colocalization are not statistically treatment. The other compound, YM201636 (33), inhibits PIK- significant. (Scale bars: 10 μm.) *P < 0.05, **P < 0.01. fyve, a kinase that converts PtdIns(3)P into PtdIns(3,5)P2 during endosome maturation. PIKfyve inhibition resulted in a similar vacuolated phenotype with highly enlarged Rab7–positive endosomes in all cells. Together, the light and electron microscopy data revealed the Electron microscopy (EM) of Cul3-depleted cells revealed the accumulation of LEs with distorted morphology upon Cul3 de- existence of enlarged vacuoles, in both HeLa and A549 cells (Fig. pletion, consistent with defects in LE maturation. 6F and Fig. S5). These vacuoles appeared empty, largely devoid of ILVs and other visible luminal content, with a diameter up to Discussion 1–1.5 μm. They were present only in a fraction of Cul3-depleted Here, we provide evidence for a previously undescribed role of cells and, thus, probably represented the aforementioned Rab7 Cul3 as a regulator of the endolysosomal pathway. Depletion of positive/fluid-filled enlarged endosomes. Cul3 resulted in the deformation of LEs and a defect in the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1118744109 Huotari et al. Downloaded by guest on September 23, 2021 transport of endocytic cargo to LYs. By tracking the fate of EGF/ EGFR and IAV, we could characterize the block and determine siControl siCul3 some of its consequences. A fi CD63 signal The early steps in the processing and traf cking of EGF and its receptor were not affected by Cul3 depletion. Upon EGF binding, HeLa A549 * EGFR was phosphorylated and ubiquitinated. Endocytic in- HeLa ternalization was normal, although somewhat more efficient than 2.5 ** 2.0 in control cells, possibly due to the increased amount of receptor. 1.5 The EGF/EGFR complexes entered EEs from which they were 1.0 sorted into LEs with a slight delay. Whether EGFR was seques- 0.5 tered into ILVs as in normal cells is not clear. Nevertheless, the A549 normalized to siControl 0.0 EGF/EGFR complexes were transported to compartments con- siControl siCul3 taining the LE/LY marker LAMP1. Because degradation of EGFR and EGF was slower than normal, these probably repre- B LysoTracker Phase Merge sented modified LEs, which failed to efficiently fuse with LYs. The fate of incoming IAV told a similar story. In Cul3-depleted cells, the virus particles were transported to LAMP1-positive vacuoles. However, uncoating of the viral capsid and transport of vRNPs to the nucleus did not occur and, as a result, the cells were siControl not infected. Importantly, the Cul3 requirement was bypassed when virus infection was artificially induced without involvement of endocytosis. Moreover, VSV, a virus that is acid-activated in the EE compartment, had no problem infecting Cul3-depleted cells, supporting the conclusion that Cul3 depletion affected mainly late endosomal compartments. siCul3 Judging by the A1 antibody staining, the HA of IAV converted to the acid-induced conformation in Cul3-depleted cells, but the vRNPs and the M1 protein remained associated with endosomal CELL BIOLOGY siControl siCul3 vacuoles, indicating that capsid uncoating and vRNP release did C D not take place. It is unclear whether this phenotype was because Vacuolated the virus failed to fuse, whether fusion was incomplete (hemi- phenotype 50 ** fusion), or whether the capsids failed to undergo disassembly after release into the cytosolic surface of endosomes. The block re- mRFP-Rab5 40 sembled the effect of amantadine, an inhibitor of the M2 ion 30 channel in the viral membrane. M2 allows acidification of the viral 20 interior before viral membrane fusion inducing a disassembly- 10 competent state in the capsid (16). Thus, in the presence of vacuolated phenotype

% of cells with EGFP-Rab7 0 amantadine, fusion takes place, but the capsids remain associated EGFP-Rab7 siControl siCul3 with endosomes and fail to release the vRNPs for nuclear import (14). Although acidic enough to convert the HA, the ionic envi- siControl siCul3 E ronment in modified LEs may not fulfill all requirements for proper M2 action. It was recently proposed that M2 has antiporter- like activity for cations such as K+ and Na+, and that this activity is required for full acidification of the IAV core (34). During maturation, endosomes undergo a number of ionic changes within the

Dextran-AF 488 lumen (35), and such changes may be used by the virus to initiate viral core acidification. A similar block in IAV infection has been F siControl siCul3 demonstrated upon inhibition of protein kinase C βII, where viruses are trapped in LEs, although the acid-mediated HA conversion has occurred (10). A requirement of an endosomal membrane potential for uncoating or membrane fusion has been proposed for other viruses, including human rhinovirus type 2 (HRV2) and Semliki forest virus (SFV) (36, 37). The defects in the late endocytic compartments in Cul3-depleted cells were reflected by a distorted morphology and aberrant Fig. 6. LE/LY morphology is distorted in Cul3-depleted cells. (A) Cul3 de- behavior of LE/LYs. The size and number was increased in some pletion results in accumulation of LE/LYs. siRNA-transfected HeLa and A549 of the cells showing a highly vacuolated phenotype with large, cells were stained with antibody against CD63 (green) and nuclei with spherical, Rab7-positive, fluid-filled endosomes observed by light DRAQ5 (blue). Images are z-projections of confocal slices. Graph shows FACS microscopy in live cells. EM studies further revealed a fraction of measurement of total CD63 immunofluorescence associated to cells. Bars Cul3-depleted cells to contain enlarged empty vacuoles virtually represent means with SEM, normalized to control siRNA from three in- devoid of ILVs. Vacuolation was comparable to cells treated with < < dependent experiments. **P 0.01, *P 0.05. (B) LysoTracker Green DND- inhibitors of PIKfyve, a known regulator of LE maturation (20), as 26 staining of live siRNA transfected HeLa cells. Some Cul3-depleted cells well as of neddylation, a crucial modification needed for Cul3- showed a highly vacuolated phenotype, as seen in the phase contrast image. dependent E3 ligase activity (32). Together, the results indicated (C) mRFP-Rab5, EGFP-Rab7, and siRNA cotransfected HeLa cells, imaged live. (D) Quantification of vacuolated phenotype from stably transfected HeLa that Cul3 is involved in the regulation of endosome maturation, cells, with inducible EGFP-Rab7 expression. Bars represent means with SEM most probably through ubiquitination. where n > 300 cells, from three independent experiments. **P < 0.01. (E) Interestingly, the phenotypes of cells treated with the protea- Live imaging of fluid phase marker Dextran-Alexa Fluor 488 loaded cells. (F) some inhibitor lactacystin have similarities to Cul3-depleted cells Thin section EM of siRNA-transfected HeLa cells. (Scale bars: A,20μm; B, C, including a delay in lysosomal degradation of transmembrane and E,10μm; F,1μm.) proteins such as EGFR (38), a block of cargo deubiquitination

Huotari et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 (39), and inhibition of IAV infection at the level of endosomes number of physiological processes as well as pathological states (40). Lactacystin-treated cells also contain enlarged, empty endo- such as infections, cancer, and neurodegeneration, deeper mosomes with EGFR trapped in the limiting membrane instead of lecular understanding of these processes provides a rewarding ILVs (38). At the same time, these cells still contain a population research direction for the future. of ILV-filled endosomes. Similarly, temperature-sensitive Ts20 mutant cells that possess a thermolabile E1 ligase also accumulate Materials and Methods acidic, hydrolase containing vacuoles, with similar characteristics Cells. A549 and HeLa cells were cultured in DMEM (Invitrogen) supplemented as observed after Cul3 depletion (41). A Cul3-based E3 ligase with 10% FCS and glutamax. could thus be responsible for the ubiquitination and proteasomal degradation of substrates involved in endosome maturation and, RNAi. All siRNAs (Microsynth/Qiagen) were reverse transfected with Lipofect- thus, ILV biogenesis. Moreover, subcellular fractionation of amine RNAiMAX (Invitrogen) for 72 h at 20 nM. Transfection efficiency was membranes revealed the presence of Cul3 on different early and controlled with Allstar death siRNA (Qiagen) and down-regulation efficiency late endosomal vesicles, suggesting that Cul3 might exert its assessed by immunoblotting or quantitative RT-PCR. The following siRNA function directly on these vesicles (Fig. S6). duplexes (from Qiagen) were used to specifically down-regulate genes: Cul3-5 Cul3-based E3 ligases recognize their substrate proteins (oligo1), Cul3-10 (oligo2), Cul1-3, c-Cbl-8, ATP6V1B2-3, HRS-5, Rab7A-5, and as “ ” “ ” through a family of adaptors bearing BTB motifs (42). The human control siRNAs either Allstar negative (Qiagen) or Scrambled (Microsynth). genome encodes ≈200 BTB proteins. Recent siRNA screens For all Cul3 siRNA experiments, oligo1 was used unless otherwise indicated. suggest a role for some of them in the endolysosomal and recycling Further details on materials and methods can be found in SI Materials pathways (43). In addition, some voltage-gated potassium chan- and Methods. nels possess BTB-like T1 domains (42), raising the possibility that ACKNOWLEDGMENTS. We thank 3-V Biosciences, Inc. for sharing their RNAi misregulation of ion channels could be a cause for endosome screening data; Chin Ha Chung, Jean Gruenberg, and Stephen Taylor for swelling upon Cul3 depletion. Clearly, the identification and reagents; Alicia Smith and Jason Mercer for critical reading of the manuscript; characterization of relevant BTB-containing proteins required for and the A.H. and M.P. group members for helpful discussions. We acknowl- maturation of LEs will be important. These findings, in turn, will edge support by the Light- and Electron Microscopy Centers at ETH Zurich. aid in the identification of the possible substrates involved. N.M.-S. and H.M. were supported by grants from the Boehringer-Ingelheim- fi Fonds. The M.P. and A.H. laboratories were funded by the European Research In conclusion, we identi ed a previously undescribed function Council, the Swiss National Science Foundation, and ETH Zurich; and for Cul3-mediated ubiquitination in endosome maturation. Con- the A.H. laboratory was supported by National Institutes of Health Grant sidering the important role of the endolysosomal pathway in a 1UO1AI074523 and a Marie Curie Initial Training Networks grant.

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