Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila.

Abstract

Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.

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Hedgehog-Regulated Ubiquitination Controls
Smoothened Trafficking and Cell Surface Expression in
Drosophila
Shuang Li1, Yongbin Chen1, Qing Shi1, Tao Yue1, Bing Wang1, Jin Jiang1,2*
1Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 2Department of
Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America
Abstract
Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo)
in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by
ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its
ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain
(SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh
inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation.
Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery
leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual b-arrestin Kurtz (Krz)
interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is
counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell
surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to
keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface
accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.
Citation: Li S, Chen Y, Shi Q, Yue T, Wang B, et al. (2012) Hedgehog-Regulated Ubiquitination Controls Smoothened Trafficking and Cell Surface Expression in
Drosophila. PLoS Biol 10(1): e1001239. doi:10.1371/journal.pbio.1001239
Academic Editor: Konrad Basler, University of Zurich, Switzerland
Received June 17, 2011; Accepted November 23, 2011; Published January 10, 2012
Copyright: ? 2012 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from NIH (GM61269) and Welch Foundation (I-1603) to J. Jiang. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: A, anterior; Avl, Avalanche; b2AR, b2-Adrenergic Receptor; CHX, cycloheximide; Ci, Cubitus interruptus; C-tail, carboxyl intracellular tail; dpp,
decapentaplegic; dsRNA, double-stranded RNA; FS, Fz2-SAID; Fz2, Frizzle 2; GPCR, G protein coupled receptor; Hh, Hedgehog; Hrs, HGF-regulated tyrosine kinase
substrate; Krz, Kurtz; mSmo, mammalian Smo; MVBs, multivesicular bodies; P, posterior; Ptc, Patched; RTK, receptor tyrosine kinase; SAID, Smo autoinhibitory
domain; Smo, Smoothened; Wg, Wingless
* E-mail: jin.jiang@utsouthwestern.edu
Introduction
Hedgehog (Hh) signaling governs cell growth and patterning in
species ranging from insects to human [1,2]. Because of its pivotal
role in embryonic development and adult tissue homeostasis,
misregulation of Hh signaling activity has been linked to many
human disorders including birth defects and cancers [1,3,4]. Hh
exerts its biological influence through a largely conserved signaling
cascade that culminates at the activation of latent transcription
factors Cubitus interruptus (Ci)/Gli [1].
The core Hh reception system consists of a 12-transmembrane
protein Patched (Ptc) that acts as the Hh receptor and a seven-
transmembrane protein Smo that acts as the Hh signal transducer
[5,6]. Hh and Ptc reciprocally regulate the subcellular localization
and active state of Smo [7–10]. In Drosophila, Hh stimulation or
loss of Ptc leads to cell surface accumulation of Smo [7,11].
Increased cell surface expression and activation of Smo are
regulated by Hh-induced and PKA/CK1-mediated phosphoryla-
tion of Smo carboxyl intracellular tail (C-tail) [12–14].
Several observations suggest that Smo cell surface expression is
controlled by endocytic trafficking. A transmission electron
microscopic study of Drosophila imaginal discs indicated that
Smo is localized primarily in the lysosome of anterior compart-
ment cells but is enriched on the plasma membrane of posterior
compartment cells [15]. In Drosophila salivary gland cells,
blocking endocytosis promotes Smo cell surface accumulation
[11]. Using antibody uptake assay in S2 cells, we have shown that
Smo reaches the cell surface but quickly internalizes in the
absence of Hh and that Hh stimulation diminishes internalized
Smo with a concomitant increase in cell surface Smo [13]. Taken
together, these observations suggest that Hh signaling may
regulate Smo cell surface expression by blocking its endocytosis
and/or promoting its recycling back to the cell surface after
internalization.
The mechanisms by which Smo endocytic trafficking and cell
surface expression are regulated have remained unknown. Smo
intracellular regions lack recognizable endosomal-lysosomal sort-
ing signals such as the NPXY and dileucine-based motifs [16].
However, many membrane receptors are internalized after
covalently modified by ubiquitination, as has been demonstrated
for receptor tyrosine kinases (RTKs) and G protein coupled
receptors (GPCRs) [17,18]. The close relationship between Smo
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and GPCRs prompted us to investigate whether Smo cell surface
expression is regulated by the ubiquitin pathway. Here we provide
both genetic and biochemical evidence that Smo trafficking and
degradation are regulated through multi-site ubiquitination of
Smo C-tail and that Hh promotes Smo cell surface expression by
inhibiting its ubiquitination. We also provide evidence that the
non-visual b-arrestin Kurtz (Krz) acts in parallel with Smo
ubiquitination to control Smo cell surface expression, and that the
deubiquitinating enzyme UBPY promotes Smo cell surface
expression by counteracting Smo ubiquitination.
Results
Inactivation of the Ubiquitin-Activating Enzyme Uba1
Leads to Smo Accumulation
In Drosophila wing discs, Smo cell surface level is low in anterior
(A) compartment cells away from the A/P boundary but is
elevated in response to Hh in A-compartment cells near the A/P
boundary or in posterior (P) compartment cells (Figure 1A) [7]. To
determine whether Smo is downregulated by the ubiquitin
pathway, we generated mutant clones for Uba1, which encodes
the only ubiquitin-activating enzyme (E1) in Drosophila [19,20]. We
employed a temperature-sensitive allele of Uba1, Uba1H33, which
behaves like a null allele at the restrictive temperature [19].
Uba1H33clones were induced at second instar larval stage (48–72 h
AEL) by FRT/FLP mediated mitotic recombination. Larva
carrying Uba1H33clones were grown at permissive temperature
(18uC) for 3 d and then shifted to non-permissive temperature
(30uC) for 24 h before dissection for immunostaining. We found
that anteriorly situated Uba1H33clones accumulated high levels of
Smo compared with neighboring wild type cells (Figure 1A–B’),
suggesting that Smo is downregulated via the ubiquitin pathway in
the absence of Hh. Immunostaining with anti-Smo antibody
before membrane permeabilization suggested that Smo was
accumulated on the cell surface in anteriorly situated Uba1H33
clones (Figure S1A–A’). A 12-h temperature shift resulted in a less
robust Smo accumulation in Uba1H33clones (Figure S1B–B’),
likely due to the perdurance of Uba1 activity. In general, Smo
elevation coincided well with Uba1 mutant clones. Intriguingly,
Uba1H33mutant cells situated in the posterior compartment also
exhibited slightly higher levels of Smo than neighboring wild type
cells (arrowhead in Figure 1B), suggesting that a fraction of Smo
still undergoes ubiquitin-mediated degradation in the presence of
Hh.
Uba1 Regulates Smo Ubiquitination and Cell Surface
Expression
To examine whether Smo is directly ubiquitinated and
whether Uba1 is responsible for this activity, we carried out a
cell-based ubiquitination assay (see Materials and Methods)
[21]. We employed RNAi and/or pharmacological inhibitor to
inactivate Uba1. S2 cells stably expressing a Myc-tagged
Smo (Myc-Smo) were treated with Uba1 or control double-
stranded RNA (dsRNA) in the absence or presence of PYR-41, a
cell permeable E1 inhibitor [22]. The efficiency of Uba1 RNAi
was confirmed by Western blot analysis of an exogenously
expressed tagged Uba1 (Figure 1C). Myc-Smo was ubiquitinated
efficiently in the absence of Uba1 inhibition (Figure 1D);
however, ubiquitination of Smo was attenuated by Uba1 RNAi
and more significantly inhibited by PYR-41 (Figure 1D). The
incomplete blockage of Smo ubiquitination by Uba1 RNAi is
likely due to partial inactivation of Uba1 by the RNAi approach.
Indeed, a combined treatment with Uba1 RNAi and PYR-41
resulted in a more complete inhibition of Smo ubiquitination
(Figure 1D).
We next applied a cell-based immunostaining assay to
determine whether Uba1 regulates Smo cell surface expression
[13]. Myc-Smo expressing cells were treated with control or Uba1
dsRNA in the absence or presence of PYR-41. Cell surface and
total Smo were visualized by immunostaining with an anti-SmoN
antibody prior to and after cell membrane permeabilization,
respectively. As shown in Figure 1E, inhibition of Uba1 either by
RNAi or PYR-41 increased the levels of Smo cell surface
expression and combined treatment resulted in more dramatic
cell surface accumulation of Smo.
Perturbation of Endocytic Machinery Leads to Smo
Accumulation and Hh Pathway Activation
Ubiquitinated membrane proteins are internalized through the
endocytic pathway and targeted to lysosome for degradation [17].
We therefore examined the effect of inactivation of endocytic
components on Smo accumulation in wing imaginal discs. We
found that Smo was accumulated in intracellular puncta in mutant
clones lacking the Drosophila homolog of HGF-regulated tyrosine
kinase substrate (Hrs) (Figure 2A–A’), a protein involved in sorting
ubiquitinated membrane proteins into multivesicular bodies
(MVBs) [23]. Of note, not all hrs mutant cells exhibited Smo
puncta. This could be due to perdurance of Hrs activity and/or
disc folding so that Smo puncta are present at different focal
planes. RNAi of other endocytic components, including Tsg101
[24], Avalanche (Avl), a Drosophila syntaxin located in early
endosomes [25], and Rab5, resulted in Smo accumulation in
anterior compartment cells distant from the A/P boundary (arrows
in Figure 2B–E), as well as Hh pathway activation as indicated by
Ci accumulation and ectopic expression of a Hh target gene
decapentaplegic (dpp) (Figure 2B–E). Taken together, these observa-
tions suggest that Smo is downregulated via the endocytic pathway
in the absence of Hh.
Author Summary
The Hedgehog (Hh) family of secreted proteins governs
cell growth and patterning in diverse species ranging from
Drosophila to human. Hh signals across the cell surface
membrane by regulating the subcellular location and
conformation of a membrane protein called Smoothened
(Smo). In Drosophila, Smo accumulates on the cell surface
in response to Hh, whereas in the absence of Hh it is
internalized and degraded. The molecular mechanisms
that control this intracellular trafficking and degradation of
Smo were unknown, but here we show that Smo is
modified by attachment of several molecules of a small
protein called ubiquitin, which tags it for internalization
and degradation within the cell. Hh inhibits this ubiquiti-
nation of Smo by inducing another modification, phos-
phorylation, of its intracellular tail by two types of protein
kinase enzymes. This loss of ubiquitination and gain of
phosphorylation causes the accumulation of Smo at the
cell surface. What’s more, we find that another protein
called Kurtz interacts with Smo and acts in parallel with the
ubiquitination process to promote internalization of Smo,
and that the deubiquitinating enzyme UBPY/USP8 coun-
teracts ubiquitination of Smo to promote its cell surface
accumulation. Our study demonstrates that reversible
ubiquitination plays a key role in regulating Smo traffic-
king to and from the cell surface and thus it provides novel
insights into the mechanism of Hh signaling from the
outside to the inside of the cell.
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Smo Is Degraded by Both Lysosome and Proteasome
Consistent with Smo being downregulated through the endocytic
pathway, treating Myc-Smo expressing S2 cells with a lysosome
inhibitor, NH4Cl, stabilized Smo (Figure 3A). Interestingly, treating
cells with a proteasome inhibitor, MG132, stabilized Smo more
dramatically than treating cells with NH4Cl (Figure 3A). Further-
more, combined treatment of cells with MG132 and NH4Cl had an
additive effect onSmostabilization(Figure 3A), suggesting that Smo
is downregulated by both lysosome- and proteasome-dependent
mechanisms. However, unlike the case of Hh stimulation or Uba1
inaction where Smo was accumulated on the cell surface,
proteasome inhibition stabilized Smo in intracellular vesicles
(Figure 3B). Double labeling with endosomal markers YFP-Rab5
(for early endosomes) or YFP-Rab7 (for late endosomes) revealed
that Smo was stabilized in Rab7 positive late endosomes after
MG132 treatment (Figure 3C). Taken together, these observations
suggest that a fraction of internalized Smo was degraded by
proteasome in the endocytic pathway before reaching to lysosome.
Hh Inhibits Smo Ubiquitination Via PKA/CK1-Mediated
Phosphorylation
Hh induces Smo cell surface accumulation both in vitro and in
vivo [7,11,13]. If Smo ubiquitination is responsible for its
internalization, Hh may increase Smo cell surface expression by
inhibiting its ubiquitination. Indeed, treating Myc-Smo stably
expressing cells with Hh-conditioned medium markedly reduced
but did not completely abolish Smo ubiquitination (Figure 4A).
Similarly, Ptc RNAi also reduced Smo ubiquitination (Figure 4B).
Our previous study demonstrated that Hh induced Smo cell
surface accumulation through PKA/CK1-mediated phosphoryla-
tion of Smo C-tail [13]. We therefore determined whether Hh
regulates Smo ubiquitination in a manner depending on Smo
phosphorylation. We found that Hh stimulation failed to inhibit
Smo ubiquitination in the presence of a PKA inhibitor H-89
(Figure 4C). On the other hand, expressing a constitutively active
PKA catalytic domain (mC*) inhibited Smo ubiquitination in the
absence of Hh (Figure 4D). To further determine whether Smo
Figure 1. Uba1 regulates Smo ubiquitination and cell surface expression. (A–B’) Low (A, B) and high (A’, B’) magnification view of wing
imaginal discs carrying Uba1H33mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. Uba1H33mutant clones are
marked by the lack of GFP staining. Arrows and arrowheads indicate anterior and posterior clones, respectively. (C) The efficiency of Uba1 RNAi was
evaluated by Western blot analysis of transfected Myc-Uba1. (D) S2 cells stably expressing a Myc-tagged Smo under the control of metallothionein
promoter were treated with Uba1 dsRNA or control (Luciferase) dsRNA in the absence or presence of the E1 inhibitor PYR41. After treatment with
MG132, cells extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with an anti-Ub antibody to
visualize ubiquitinated Smo (top) or anti-Myc antibody to visualize Myc-Smo (bottom). Loading was normalized by the amount of Myc-Smo
monomer. IP, immunoprecipitation; IB, immunoblot. (E) S2 cells stably expressing Myc-Smo were treated as in (D). Cells were immunostained with
anti-SmoN antibody before membrane permeabilization to visualize cell surface Smo (top panels) or after membrane permeabilization to examine
the total Smo (bottom panels). Quantification of cell surface and total Smo levels was shown (20 cells for each condition). The numbers indicate the
ratio of cell surface Smo signal versus total Smo signal.
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ubiquitination is regulated by PKA/CK1-mediated phosphoryla-
tion of its C-tail, S2 cells were transfected with Myc-tagged wild
type Smo, a phosphorylation deficient form of Smo (SmoSA) with
three PKA sites (S667, S687, and S740) mutated to Ala, or a
phospho-mimetic form of Smo (SmoSD) with three PKA/CK1
clusters mutated to Asp [13], treated without or with Hh
conditioned medium, and followed by the ubiquitination assay
described above. As shown in Figure 4E, Hh inhibited the
ubiquitination of Myc-Smo but did not significantly affect the
ubiquitination of Myc-SmoSA. Furthermore, the phospho-mimetic
Smo mutant, SmoSD, exhibited diminished ubiquitination and its
residual ubiquitination was further reduced by Hh treatment
(Figure 4E–F). These results support the notion that Hh-induced
phosphorylation by PKA/CK1 inhibits Smo ubiquitination,
leading to its cell surface accumulation.
The SAID Domain Promotes Smo Ubiquitination and
Endocytosis
Our previous study revealed that the Smo autoinhibitory
domain (SAID) inhibits Smo activity in part by preventing Smo
cell surface expression because a Smo variant lacking the SAID
domain (SmoD661–818or SmoDSAID) accumulated on the cell
surface in the absence of Hh stimulation [8]. To determine
whether the SAID domain regulates Smo ubiquitination, we
examined the ubiquitin status of a Myc-tagged SmoDSAID(Myc-
SmoDSAID). As shown in Figure 4G, deleting the SAID domain
diminished Smo ubiquitination, and the residual ubiquitination of
Myc-SmoDSAIDwas further reduced by Hh treatment.
To determine whether the SAID domain suffices to promote
ubiquitination and internalization of a heterologous membrane
protein, we fused it to the C-terminus of the Wingless (Wg)
receptor Frizzle 2 (Fz2) to construct Fz2-SAID chimeric protein
(FS). When expressed in S2 cells, CFP-tagged Fz2 (CFP-Fz2) was
largely accumulated on the cell surface with a small fraction
internalized and colocalized with the endosomal marker Rab5
(Figure 5A–A’’). In contrast, CFP-FS was barely detectable on the
cell surface but largely accumulated in Rab5-positive endosomes
(Figure 5B–B’’), suggesting that SAID can promote endocytosis of
Fz2. Introducing the phosphorylation-mimetic mutation to the
SAID domain of FS (CFP-FS-SD) reduced its endocytosis
(Figure 5C–C’’), whereas the chimeric protein carrying a
phosphorylation deficient form of SAID (CFP-FS-SA) was
internalized as efficiently as CFP-FS (Figure 5D–D’’). In addition,
we found that adding the phosphorylation-deficient form but not
the phospho-mimetic form of SAID to Fz2 promotes the
ubiquitination of the corresponding chimeric protein (Figure 5E).
Taken together, these observations suggest that the SAID domain
suffices to promote ubiquitination and internalization of a
membrane protein in a manner inhibited by phosphorylation.
Combined with our earlier work [8], it seems that the SAID
domain autonomously regulates ubiquitination independent of the
C-terminal negatively charged region.
Smo Is Ubiquitinated at Multiple Lysine Residues
If Smo ubiquitination is responsible for its internalization and
degradation, one would expect that ubiquitination-deficient Smo
variants should be stabilized and accumulated on the cell surface.
We therefore attempted to identify Lys residues responsible for
Smo ubiquitination. In general, ubiquitin acceptor sites lack a
strict consensus and target proteins can be ubiquitinated at
multiple Lys residues. Smo C-tail and intracellular loops contain a
total of 49 Lys residues, many of which may serve as ubiquitin
acceptor sites, making it difficult to generate Smo variants devoid
of ubiquitination. As deleting the SAID domain diminished Smo
ubiquitination (Figure 4G), we speculated that this region might
contain Lys residues critical for Smo ubiquitination. There are a
total of 13 Lys residues between aa 661 and aa 818. We therefore
constructed SmoK6Rwith K665, K695, K700, K702, K710, and
K733 mutated to Arg; SmoK7Rwith K752, K753, K762, K772,
K773, K782, and K801 mutated to Arg; and SmoK13Rwith all the
Figure 2. Smo accumulates in cells defective in the endocytic machinery. (A–A’) Low (A) and high (A’) magnification view of a wing imaginal
disc carrying hrs mutant clones and immunostained with anti-SmoN (red) and anti-GFP (green) antibodies. hrs mutant clones are marked by the lack
of GFP staining (arrows). (B–E) A wild type wing disc (C) or wing discs expressing UAS-Tsg101-RNAi (B), UAS-Avl-RNAi (D), or UAS-Rab5-RNAi (E) with the
MS1096 Gal4 driver were immunostained to show the expression of Smo (red), Ci (green), and dpp-lacZ (blue). Arrows indicate Smo and Ci
accumulation (B, D, E) as well as ectopic dpp-lacZ expression (D, E) in A-compartment cells situated distantly from the A/P boundary. Of note, UAS-
Dicer2 was coexpressed with UAS-Tsg101-RNAi and UAS-Avl-RNAi to enhance the RNAi effect.
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13 Lys residues mutated to Arg. Using the cell-based ubiquitina-
tion assay described above, we found that both Myc-SmoK6Rand
Myc-SmoK7Rexhibited reduced ubiquitination compared with
Myc-Smo (Figure 6A). The combined mutations (K13R) resulted
in a more dramatic reduction in Smo ubiquitination (Figure 6A),
suggesting that Smo is ubiquitinated at multiple Lys residues
between aa 661 and aa 818. In addition, the residual ubiquitina-
tion of Myc-SmoK13Rsuggests that Smo is also ubiquitinated at
one or more Lys residues outside the SAID domain.
SmoK13RExhibits Increased Stability and Cell Surface
Expression
We next determined whether the K13R mutation affects Smo
stability and cell surface expression. Myc-Smo and Myc-SmoK13R
expression constructs were transfected into S2 cells together with a
Myc-CFP expression construct as an internal control. The levels of
Myc-Smo and Myc-SmoK13Rwere monitored at different time
points after treatment with the protein synthesis inhibitor,
cycloheximide (CHX). As shown in Figure 6B, Myc-SmoK13R
exhibited increased half-life compared with Myc-Smo, suggesting
that inhibition of Smo ubiquitination leads to its stabilization. We
also measured the steady state levels of Myc-Smo and Myc-
SmoK13Rin the absence or presence of MG132 and/or NH4Cl.
While Myc-Smo was stabilized by both MG132 and NH4Cl, Myc-
SmoK13Rwas stabilized by NH4Cl but insensitive to MG132
treatment (Figure 6C), suggesting that inhibition of Smo
ubiquitination blocks its degradation by proteasome.
To determine whether inhibition of Smo ubiquitination leads to
its cell surface accumulation, S2 cells were transfected with Myc-
Smo or Myc-SmoK13Rexpression construct, followed by treatment
with or without Hh-conditioned medium. Cell surface and total
Smo were monitored by immunostaining with the anti-SmoN
antibody before and after cell permeabilization, respectively. As
shown in Figure 6D, Myc-SmoK13Rexhibited higher basal level of
cell surface expression than Myc-Smo; however, the level of cell
surface Myc-SmoK13Rin the absence of Hh was still lower than
that of Myc-Smo or Myc-SmoK13Rin the presence of Hh
(Figure 6D). Thus, although SmoK13Rexhibits increased stability
and cell surface expression, it is still internalized and degraded by
lysosome and can be further stabilized by Hh.
To determine whether the K13R mutation affects Smo stability
in vivo, we generated transgenic flies expressing either UAS-Myc-
Smo or UAS-Myc-SmoK13Rfrom the same genetic locus using the
phiC31 integration system to ensure similar expression level from
different constructs [26]. We used the wing specific Gal4 driver
MS1096 coupled with tub-Gal80tsto drive a pulse of UAS-Myc-Smo
or UAS-Myc-SmoK13Rexpression by shifting late third instar larvae
to the non-permissive temperature for 12 h. After chasing for
Figure 3. Smo is stabilized by both lysosome and proteasome inhibitors. (A) S2 cells stably expressing Myc-Smo were treated with MG132
and/or NH4Cl alone or in combination, followed by Western blot analysis with an anti-Myc antibody. (B) S2 cells stably expressing Myc-Smo treated
with or without MG132 and/or Hh-conditioned medium were immunostained with anti-SmoN antibody before membrane permeabilization to
visualize cell surface Smo (top panels) or after membrane permeabilization to examine the total Smo (bottom panels). MG132 treatment stabilized
Smo in intracellular vesicles whereas Hh treatment led to cell surface accumulation of Smo. (C) Myc-Smo expressing S2 cells were transfected with
YFP tagged Rab5 or Rab7, treated with or without MG132 and immunostained to show the expression of Myc-Smo (green) and Rab5/Rab7 (red).
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