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Molecular Cell

Article

Failure of Amino Acid HomeostasisCauses Cell Death following Proteasome InhibitionAmila Suraweera,1 Christian Munch,2 Ariane Hanssum,1 and Anne Bertolotti1,*1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK2Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

*Correspondence: [email protected]://dx.doi.org/10.1016/j.molcel.2012.08.003

SUMMARY

The ubiquitin-proteasome system targets manycellular proteins for degradation and thereby con-trols most cellular processes. Although it is wellestablished that proteasome inhibition is lethal, theunderlying mechanism is unknown. Here, we showthat proteasome inhibition results in a lethal aminoacid shortage. In yeast, mammalian cells, and flies,the deleterious consequences of proteasome inhibi-tion are rescued by amino acid supplementation. Inall three systems, this rescuing effect occurs withoutnoticeable changes in the levels of proteasome sub-strates. In mammalian cells, the amino acid scarcityresulting from proteasome inhibition is the signalthat causes induction of both the integrated stressresponse and autophagy, in an unsuccessful attemptto replenish the pool of intracellular amino acids.These results reveal that cells can tolerate proteinwaste, but not the amino acid scarcity resultingfrom proteasome inhibition.

INTRODUCTION

The controlled degradation of proteins by the proteasome is

essential in all cells and impairment of the ubiquitin-proteasome

system contributes to many pathological conditions (Hershko

and Ciechanover, 1998; Finley, 2009). Although the proteasome

inhibitor Bortezomib is used for the treatment of multiple

myeloma (Hideshima et al., 2001; Goldberg, 2007; Navon and

Ciechanover, 2009), it remains unclear how proteasome failure

kills cells.

Because the ubiquitin-proteasome system controls the degra-

dation of a large number of cellular proteins including short-lived,

regulatory, and damaged or misfolded proteins (Hershko and

Ciechanover, 1998; Schwartz and Ciechanover, 2009), it has

been assumed that accumulation of no-longer needed proteins

underlies the toxicity of proteasome inhibition. However, how

such proteins become harmful to cells and organisms remains

unclear. Failure to degrade key regulatory proteins could also

perturb cellular function. The transcription regulator NF-kB is

one such regulatory protein activated by proteasome cleavage

(Palombella et al., 1994). NF-kB is constitutively activated in

multiple myeloma and thus was thought to be a prominent

242 Molecular Cell 48, 242–253, October 26, 2012 ª2012 Elsevier Inc

target of proteasome inhibitors (Schwartz and Ciechanover,

2009). However, as selective inhibition of NF-kB does not

recapitulate the cytotoxic effects of proteasome inhibition

(Hideshima et al., 2002), alternative mechanisms need to be

considered.

The function of the proteasome in protein waste disposal

has been extensively studied (Hershko and Ciechanover, 1998;

Finley, 2009). In addition to this well-described function, the

proteasome also recycles amino acids. The importance of this

aspect of proteasomal degradation for normal cell and organ-

ismal function is unclear. In contrast, recycling amino acids is

a well-established function of autophagy in starving cells (Naka-

togawa et al., 2009). Under conditions of amino acid starvation,

both autophagy and proteasomal degradation are required to

maintain adequate amino acid levels for sustaining protein

synthesis (Onodera and Ohsumi, 2005; Vabulas and Hartl,

2005). Despite the established importance of protein recycling

under conditions of severe nutrient deficiency, it is unclear

whether protein degradation contributes an important fraction

of the supply of amino acids under physiological conditions.

Because the mechanisms underlying the toxicity resulting

from proteasome inhibition are unclear, and as it is unknown

whether protein degradation contributes an essential fraction

of the intracellular amino acid pool under normal conditions,

we examined whether the lethality resulting from proteasome

inhibition is mediated by perturbation of amino acid homeo-

stasis. In three model systems—yeast, mammalian cells, and

Drosophila—we find that proteasome inhibition causes a lethal

shortage of amino acids and the lethality resulting from protea-

some inhibition is rescued upon amino acid supplementation,

without any detectable decrease in the levels of proteasome

substrates in proteasome-inhibited cells.

RESULTS

Failure of Amino Acid Homeostasis Causes Cell Deathfollowing Proteasome Inhibition in YeastWe first assessed whether amino acid homeostasis was altered

following proteasome impairment in yeast. As expected (Ghislain

et al., 1993), exposure to 37�C for 20 hr was lethal to the yeast

proteasome mutant cim3-1, harboring a thermosensitive muta-

tion in the regulatory subunit Rpt6 (Figure S1A available online).

We next monitored the levels of free amino acids in yeast cells

following proteasome inhibition. Exponentially growing yeast

cells were inoculated at an optical density of 0.2 and grown at

30�C in the standard rich medium yeast peptone dextrose

.

mailto:[email protected]

http://dx.doi.org/10.1016/j.molcel.2012.08.003

H

Ura3-3(GFP)

Ponceau S

cim3-1

25 °

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37 °

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A

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100

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35S-methionine

Coomassie

AA - + - + - + - +WT cim3-1WT cim3-1

30 °C 37 °C

label WT/ cim3-1 100 19 88 %

C

dead cells

Fluorescence intensity Propidium iodide

Cel

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ber

G

4 hours 6 hours

WT

cim

3-1

Ponceau S

0 hours

WT cim3-1 WT cim3-1

30 °

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30 °

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37 °C +/- AA

2 d at 30 °C

plate

1

2

3

+ AA

cim3-1

23 h at 38 °C24 h at 30 °C

48 h at 30 °C

+ AA

pre1-1

23 h at 37 °C25 h at 30 °C

48 h at 30 °C

E FD

CLB2

Ponceau

30 °

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30 °

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30 °

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0 hours 4 hours

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37 °

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37 °

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+ A

A

37 °

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h

37 °

C 1

h +

AA

Figure 1. Amino Acid Insufficiency Mediates the Lethality Resulting from Proteasome Inhibition in Yeast Cells

(A) Changes in the content of cellular free amino acids in cim3-1 or wild-type (WT) cells after 5 hr at 37�C in rich medium (YPD). Data are means ± SD (n = 3),

normalized to cell number. Significance of all values in wild-type versus mutant cells: p% 0.001. Cells were in exponential growth at 30�C before the temperature

switch. Asx: Asn, Asp. Glx: Glu, Gln.

(B) Cell death monitored by flow cytometry analysis of propidium iodide labeled yeast cells after 21 hr at 37�C, in the presence of 40 g/l casamino acids (AA)

where indicated.

(C) Viability of cim3-1 cells exposed to 37�C for 21 hr in liquid cultures (YPD), in the presence of 40 g/l casamino acids (AA) where indicated or kept at 4�C,assessed by spotting serial dilutions on rich media and incubation at 30�C for 2 days.

(D and E) Viability of cim3-1 and pre1-1 cells on YPD plates exposed to the indicated temperatures for the indicated times, with or without casamino acids

(AA, 40 g/l). A total of 2,000 cells were plated in each condition.

(F) Polyubiquitin immunoblots (Ubn) from lysates of wild-type or cim3-1 cells, cultured as indicated. Ponceau staining of the membrane.

(G) Immunoblots of GFP-tagged Ura3-3 from lysates of the indicated cells treated with cycloheximide as in Lewis and Pelham (2009) and cultured as indicated.

(H) Accumulation of Cyclin B2 (CLB2) in cim3-1 cells at 37�C. CLB2 immunoblots from lysates of wild-type or cim3-1 cells, grown in rich medium with or without

casamino acids (AA, 40 g/l) for the indicated times at 30�C or 37�C. Ponceau staining of the membrane prior to probing with antibodies.

(I) Autoradiogram of [35S]methionine-labeled proteins in lysates resolved by NuPage after a 8 min pulse labeling of exponentially growing cells cultured as

indicated and quantification of labeled proteins. Lower panel is a photomicrograph of the Coomassie-stained gel. Representative results of at least three

independent experiments are shown.

See also Figure S1.

Molecular Cell

Proteasome Failure Kills Cells by Starvation

(YPD) to insure that the yeast cells were replete with nutrients.

Five hours after the switch to 37�C, the levels of most amino

acids increased in wild-type cells (Figure 1A). This is likely the

Mol

result of the heat shock, which is known to attenuate protein

synthesis (Richter et al., 2010). Surprisingly, however, the levels

of most free amino acids decreased in cim3-1 cells after 5 hr

ecular Cell 48, 242–253, October 26, 2012 ª2012 Elsevier Inc. 243

Molecular Cell


at 37�C (Figure 1A). Unlike wild-type cells, cim3-1 cells did not

grow at 37�C (Figure S1B), indicating that the decrease in intra-

cellular amino acids in cim3-1 cells was not due to an exhaustion

of the nutrient supply from the media as a result of cell growth.

Thus, inhibition of proteasomal degradation, under conditions

of nutrient sufficiency, decreases the levels of intracellular amino

acid in yeast cells.

We then investigated whether increasing the supply of amino

acids could rescue cim3-1 cells. Exponentially growing wild-

type or cim3-1 yeast cells were inoculated at an optical density

of 0.2 and cultured in the standard rich medium YPD for 21 hr,

labeled with propidium iodide to reveal dead cells, and analyzed

by flow cytometry. Unlike wild-type cells, the vast majority of

cim3-1 cells died after 21 hr of culture at 37�C in rich media

(Figure 1B). supplementation with amino acids prevented the

death of the cim3-1 cells exposed to 37�C (Figure 1B). To deter-

mine whether such cells were viable, they were spotted on rich

plates and grown at the permissive temperature. Unlike the non-

supplemented cim3-1 cells, cim3-1 cells that had been exposed

to 37�C in the presence of additional amino acids were viable

and gave rise to the same number of colonies as wild-type cells

at 30�C (Figure 1C). This revealed that amino acid supplementa-

tion prevents death of proteasome-inhibited yeast cells. This

rescuing effect occurs without promoting cell growth because

the number of viable cells exposed to 37�C in the presence of

amino acids equaled the number of cells prior exposure to

37�C (Figure 1C, compare lane 1 to lane 3). Supplementation

with amino acids also protected cim3-1 cells exposed to 37�Con plates (Figure 1D), as well as the proteasome mutant pre1-1

cells, harboring a thermosensitive mutation in the b4 subunit of

the 20S proteasome (Heinemeyer et al., 1991) (Figure 1E), but

not the ndc80-1mutant cells (Figure S1C), which harbors a ther-

mosensitive mutation unrelated to the proteasome, in a spindle

body component (Wigge et al., 1998).

Although rescuing viability, addition of amino acids did not

noticeably decrease the levels of polyubiquitinated proteins

(Figure 1F) or the unstable GFP-tagged Ura3-3 (Lewis and

Pelham, 2009) that accumulated in cim3-1 cells at 37�C (Fig-

ure 1G). Thus, amino acid supplementation rescues from the

lethality resulting from proteasome inhibition without promoting

a recovery of proteasome function.

Proteasomal degradation is required for progression through

the cell cycle (Hershko, 2005) and at nonpermissive temperature,

cim3-1 cells stop dividing (Ghislain et al., 1993) and (Figure S1B)

and accumulate cyclins, such as CLB2 (Ghislain et al., 1993).

Although rescuing the viability of proteasome-inhibited cells,

amino acids supplementation did not alleviate the growth arrest

resulting from proteasome inhibition (Figure S1B). Consistently,

the levels of CLB2 were indistinguishable in cim3-1 cells at

37�C with or without amino acids supplementation (Figure 1H).

This confirmed that amino acids did not rescue proteasomal

degradation or the resulting growth impairment in proteasome

inhibited cells, while rescuing viability.

We next examined if the marked decrease in amino acid noted

in cim3-1 cells impacted on protein synthesis. Note that, as ex-

pected (Richter et al., 2010), heat shock reduced protein

synthesis in wild-type cells (Figure 1I). Strikingly, we found that

in cim3-1 cells, protein synthesis at 37�C was reduced to 19%


of the wild-type cells exposed to the same temperature (Fig-

ure 1I). Supplementation with amino acids rescued translation

rates in the cim3-1 cells to nearly wild-type levels (Figure 1I).

Amino Acid Supplementation Rescues theDeleterious Consequences of Proteasome Inhibitionin Mammalian CellsBecause proteasome function is conserved in evolution, we next

examined whether inhibition of the proteasome altered amino

acid homeostasis in mammalian cells. Treatment of mammalian

NIH 3T3 cells with the proteasome inhibitorsMG-132 or Bortezo-

mib decreased the levels of the amino acids asparagine/aspar-

tate and cysteine, whereas the levels of other amino acids

remained largely unchanged or only marginally increased (Fig-

ure S2A). To address whether the decrease in cysteine and

asparagine/aspartate were detrimental to cells following protea-

some inhibition, we examined whether supplementation with

these amino acids affected cell viability of proteasome-inhibited

cells. Amino acids were added after a transient treatment with

the proteasome inhibitor to ensure that the amino acids did not

interfere with the proteasome inhibitors. Addition of cysteine

following treatment with MG-132 for 8 hr dramatically increased

cell survival and markedly reduced apoptosis in a dose-depen-

dent manner, in contrast to other amino acids, the reducing

agent b-mercaptoethanol, or the antioxidant ascorbic acid

(Figures S2B and S2C). Cysteine also markedly increased cell

viability following Bortezomib treatment (Figure S2D). Aspara-

gine further increased viability, when added together with

cysteine following an 8 hr MG-132 treatment, whereas it only

had a minor effect on its own (Figure 2A). It may not be a coinci-

dence that both cysteine and asparagine are conditionally

essential amino acids (Reeds, 2000), absent in the Dulbecco’s

modified Eagle’s medium (DMEM). Although rescuing the

viability of proteasome-inhibited cells, amino acid supplementa-

tion had no significant effect on the proliferation of untreated

cells (Figure S2E) and neither cysteine nor asparagine detectably

altered proteasome activity (Figure S2F). Thus, supplementation

with the critical amino acids depleted upon proteasome inhibi-

tion rescued survival of �90% of proteasome-impaired cells,

without rescuing proteasome activity.

To dissect the mechanism underlying the rescued viability of

proteasome-inhibited cells by amino acid supplementation, we

next focused on cysteine addition as it had a potent effect. We

monitored the abundance of polyubiquitinated proteins as well

as the proteasome reporter substrates UbG76V-GFP, ZsGreen-

ODC, and GFP-CL1 (Dantuma et al., 2000; Hoyt et al., 2005;

Gilon et al., 1998; Bence et al., 2001). Although markedly

increasing the survival of three different cell lines following

proteasome inhibition (Figure S2G), addition of cysteine did not

reduce the abundance of either polyubiquitinated proteins (Fig-

ure 2B) or the three different rapidly degraded, proteasome

reporter-substrates UbG76V-GFP, ZsGreen-ODC, and GFP-CL1

(Figure 2C). Similarly, cells accumulated indistinguishable levels

of the proteasome reporter substrate ZsGreen-ODC when

treated with MG-132 alone or together with 1 mM cysteine

(Figure 2D). Addition of cysteine together with MG-132 also

rescued the viability of proteasome-inhibited cells (Figure S2G).

These results demonstrate that cysteine did not antagonize

.

A

Asn (mM) - 10 - 2.5 5 10

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2 days

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97

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- Cys + Cys

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ZsGreen-ODC 293T

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ity

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n.s.

n.s.

Tubulin

Cys - - - +

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97

37

54

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MG-132 6 h +/- Cys 6 h

UbG76V-GFP NIH-3T3

Flu

ores

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****

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MG-132 �� Cys ��

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C

Figure 2. Failure of Amino Acid Homeostasis Causes Cell Death following Proteasome Inhibition

(A) Viability of NIH 3T3 cells, assessed by the reduction of WST-8 into formazan, after 2 days of growth, following treatment with 10 mM MG-132 for 8 hr and a

20 hr washout in regular medium, with or without cysteine (Cys) and/or asparagine (Asn). Data are means ± SD (n = 4). *p% 0.01, **p% 0.001, and ***p% 0.0001.

n.s., not significant.

(B) Polyubiquitin (Ubn) and tubulin immunoblots of lysates from NIH 3T3 cells exposed to 10 mM MG-132 treatment for 6 hr, followed by a washout in regular

medium, with or without 1 mM Cys supplementation, for the indicated time.

(C) Fluorescence of proteasome reporter-substrates UbG76V-GFP (Dantuma et al., 2000), ZsGreen-ODC (Hoyt et al., 2005), and GFP-CL1 (Gilon et al., 1998;

Bence et al., 2001) in NIH 3T3 or 293T cells, following treatment with 10 mM MG-132 and a washout for the indicated time, with or without Cys. Data are

means ± SD (n = 3). *p % 0.01, **p % 0.001, and ***p % 0.0001. n.s., not significant.

(D) Fluorescence of proteasome reporter-substrates ZsGreen-ODC (Hoyt et al., 2005) following treatment with 10 mMMG-132 either alone or together with 1 mM

Cys where indicated.

(E) Polyubiquitin (Ubn), tubulin, and NF-kB (p105 and p50) immunoblots of lysates of 293T cells overexpressing p105 and treated with 10 mM MG-132 for 8 hr,

either alone or together with 1 mM Cys, where indicated. Representative results of at least three independent experiments are shown.

See also Figure S2.

Molecular Cell


Molecular Cell 48, 242–253, October 26, 2012 ª2012 Elsevier Inc. 245

Molecular Cell


MG-132, while rescuing viability of cells following proteasome

inhibition.

The proteasome activates the transcription regulator NF-kB

by proteolytic cleavage of the precursor p105 into p50

(Palombella et al., 1994). Although rescuing cell viability (Fig-

ure 2A), cysteine supplementation did not reduce the impairment

of the proteasome-mediated processing of the transcription

regulator NF-kB, p105 into p50, in cells treated with MG-132

for 8 hr (Figure 2E). These results reveal that while rescuing

from the lethality resulting from proteasome inhibition, supple-

mentation with an amino acid selectively depleted following

proteasome inhibition did not decrease the levels of polyubiqui-

tinated proteins, proteasome reporter substrates and the impair-

ment of NF-kB processing in proteasome-inhibited cells. Thus,

cells can tolerate proteasome inhibition but not the resulting

amino acid insufficiency.

The Integrated StressResponse Is Inducedby theAminoAcid Shortage Resulting from Proteasome InhibitionProteasome inhibition, like many different stresses, induces the

integrated stress response (ISR) (Jiang and Wek, 2005).

However, the signal responsible for the ISR induction in protea-

some-inhibited cells is unknown. The ISR is an adaptive

response to many forms of stresses, which converge into phos-

phorylating the a subunit of translation initiation factor 2 (eIF2a)

thereby reprogramming protein synthesis. When eIF2a is phos-

phorylated, general protein synthesis is attenuated, whereas

ATF4 is selectively translated (Harding et al., 2003). ATF4 is a

transcription factor, which controls expression of genes

involved in amino acid import and biosynthesis (Harding et al.,

2003), as well as the transcription factor CHOP, which in turn

induces GADD34 (Figure 3A). GADD34 is a regulatory subunit

of the serine/threonine phosphatase PP1, which recruits PP1

in stressed cells to dephosphorylate eIF2a and terminates stress

signaling (Novoa et al., 2003). Having found that proteasome

inhibition caused a lethal shortage in both cysteine and aspara-

gine, and because genes involved in the biosynthesis of cysteine

and asparagine are prominent targets of the ISR (Harding et al.,

2003), we examined whether amino acid scarcity may be the

signal that induces the ISR in proteasome inhibited cells (Fig-

ure 3A). If so, amino acid supplementation should attenuate

ISR signaling in response to proteasome inhibition. In the

absence of MG-132, the ISR was not induced, confirming that

untreated cells were not stressed (Figure 3B). As expected

(Jiang and Wek, 2005), MG-132 induced the ISR, and this is

manifested by the phosphorylation of eIF2a, expression of

ATF4 and the pro-death proteins CHOP and GADD34 (Fig-

ure 3B). Strikingly, the levels of CHOP and GADD34 remained

elevated in the MG-132 cells 6 hr after the removal of the protea-

some inhibitor, in agreement with the finding that such cells

were committed to die (Figure 2A). In contrast, in cells supple-

mented with cysteine, a marked decrease in the levels of

CHOP and GADD34 was detected 4–6 hr following the cysteine

washout (Figure 3B), in good agreement with the cytoprotective

effect of cysteine upon proteasome inhibition (Figure 2A). When

added together with MG-132, cysteine virtually abrogated the

induction of the ISR resulting from proteasome inhibition,

without altering the levels of polyubiquitinated proteins (Fig-


ure S3). This reveals that the amino acid shortage resulting

from proteasome inhibition is the signal that induces the ISR in

proteasome-inhibited cells.

To confirm that CHOP-mediated cell death following protea-

some inhibition (Jiang andWek, 2005), wemonitored the survival

of wild-type and CHOP�/� cells (Marciniak et al., 2004)

following proteasome inhibition. Genetic ablation of CHOP

markedly enhanced survival of proteasome-inhibited cells (Fig-

ure 3C). The decreased CHOP levels in NIH 3T3 cells supple-

mented with cysteine following proteasome inhibition could be

due to an enhanced degradation or a decreased synthesis. We

found that addition of cysteine did not rescue proteasomal

degradation (Figures 2B–2E) and the stability of both GADD34

and CHOP were indistinguishable in cells that had been supple-

mented or not with cysteine, following proteasome inhibition

(Figure 3D). Thus, the low levels of CHOP in proteasome-

inhibited cells supplemented with cysteine did not reflect an

increased degradation but rather, a decreased synthesis of this

stress protein, as a result of the dephosphorylation of eIF2a

following cysteine supplementation (Figure 2B).

The marked attenuation of eIF2a phosphorylation by cysteine

supplementation in proteasome-inhibited cells suggested that

the supply of cysteine might be rate-limiting for global protein

synthesis in proteasome-inhibited cells. If so, cysteine supple-

mentation may rescue global protein synthesis while attenuating

translation of stressed proteins such as CHOP. We next tested

this hypothesis. As expected (Jiang andWek, 2005), proteasome

inhibition caused a pronounced inhibition of protein synthesis

(Figure 3E). Remarkably, addition of 1 mM cysteine largely pre-

vented the translation attenuation resulting form proteasome

inhibition (Figure 3E). GCN2 is the eIF2a kinase that mediates

the ISR induction in response to amino acid deprivation (Harding

et al., 2000; Zhang et al., 2002). Genetic ablation of GCN2 virtu-

ally abolished eIF2a phosphorylation upon proteasome inhibition

(Figure 3F). Furthermore, activated and phosphorylated GCN2

was detected in cells deprived of leucine or MG-132-treated

cells, and addition of cysteine markedly reduced GCN2 phos-

phorylation upon proteasome inhibition (Figure 3G). This estab-

lishes that inhibition of proteasomal degradation causes a

shortage of critical amino acids that induce the ISR via GCN2.

To confirm that the attenuation of eIF2a phosphorylation by

cysteine in proteasome inhibited cells is due to the replenish-

ment of this otherwise limiting amino acid, as opposed to a

suppression of ISR signaling by an indirect effect, we tested

whether cysteine affected eIF2a phosphorylation induced by

ultraviolet (UV) irradiation, a different stress than amino acid limi-

tation (Deng et al., 2002). We found that the induction of eIF2a

phosphorylation by UV was not reduced by supplementation

with cysteine (Figure 3H). Thus, cysteine supplementation selec-

tively alleviates eIF2a phosphorylation in proteasome-inhibited

cells, but not in UV-irradiated cells.

Amino Acid Scarcity in Proteasome-Inhibited CellsSignals the Induction of AutophagyAlthough polyubiquitination is the canonical signal to target

proteins for proteasomal degradation, recent studies have

revealed that polyubiquitinated proteins can also be degraded

by autophagy (Kirkin et al., 2009). Autophagy is also induced

.

A B

C D

E F

G H

Figure 3. Amino Acid Supplementation Alle-

viates the Induction of the Integrated Stress

Response upon Proteasome Inhibition

(A) Scheme depicting the integrated stress

response induction upon proteasome inhibition.

The signal responsible for eIF2a phosphorylation

(P-eIF2a) upon proteasome inhibition is unknown.

(B) Polyubiquitin (Ubn), GADD34, ATF4, phos-

phorylated eIF2a (P-eIF2a), eIF2a, and CHOP

immunoblots of lysates from NIH 3T3 cells treated

with 10 mMMG-132 for 6 hr followed by a washout

in the presence of 1 mM cysteine (Cys) for the

indicated time.

(C) Viability of wild-type and CHOP�/� mouse

embryonic fibroblasts, assessed by the reduction

of WST-8 into formazan, after 2 days of growth,

following treatment with 10 mM MG-132 for the

indicated time and a washout in regular medium.

Data are means ± SD (n = 4), normalized to cell

number. *p % 0.01, ***p % 0.0001.

(D) GADD34, CHOP, and tubulin immunoblots of

lysates from NIH 3T3 cells treated with 10 mMMG-

132 for 4 hr followed by a washout in the presence

of cycloheximide (CHX, 100 mg/ml), with or without

1 mM Cys for the indicated time.

(E) Autoradiogram of [35S]methionine-labeled

proteins in lysates resolved by NuPage after a

8 min pulse labeling of exponentially growing cells

cultured as indicated. Quantification are presented

below each lane. Lower panel is a photomicro-

graph of the Coomassie-stained gel.

(F) eIF2a immunoblots of lysates from GCN2�/�or wild-type mouse embryonic fibroblasts treated

with 10 mM MG-132 for the indicated time.

(G) Immunoblots of GCN2 immunoprecipitated

from lysates of cells either left untreated, leucine-

deprived (-Leu), or treated with 10 mM MG-132,

with or without 1 mM Cys. The top panel was

probed with antisera against the phosphorylated

GCN2 and the bottom panel with an antibody

recognizing both the phosphorylated and the

nonphosphorylated GCN2.

(H) Immunoblots of lysates from NIH 3T3 cells

following a dose response analysis of UV irradia-

tion, in the presence or in absence of 1 mM

cysteine (Cys). Representative results of at least

three independent experiments are shown.

See also Figure S3.

Molecular Cell


when proteasomal degradation is inhibited (Rideout et al., 2004;

Iwata et al., 2005) and alleviates the toxicity resulting from pro-

teasome inhibition (Korolchuk et al., 2010; Pandey et al., 2007).

However, the mechanisms underlying autophagy induction

when proteasomal degradation is compromised are unknown.

As expected (Korolchuk et al., 2010), MG-132 increased the

number of GFP-LC3 labeled autophagosomes in HeLa cells

stably expressing GFP-LC3 (Figures 4A and 4B). Strikingly,

cysteine supplementation markedly decreased the number of

fluorescent puncta in proteasome-inhibited cells (Figures 4A

and 4B). In contrast, supplementation with other amino acids,

reducing agents or antioxidants did not noticeably reduced the

number of GFP-LC3 puncta in proteasome inhibited cells (Fig-

Mol

ure S4). Cysteine is not a generic suppressor of autophagosome

accumulation because cysteine did not decrease the number of

GFP-LC3 puncta that accumulated in cells treated with Bafilo-

mycin A1 (Figures 4A and 4B), an inhibitor of autophagosome-

lysosome fusion (Mizushima et al., 2010). MG-132 treatment

also increased the levels of LC3-II (Figures 4C and 4D) but this

was markedly decreased in the presence of cysteine (Figures

4C and 4D). These results establish that the decrease in a critical

amino acid is the signal that induces autophagy when proteaso-

mal degradation is inhibited. To assess whether induction of

autophagy contributed to protect cells from proteasome-inhibi-

tion, we compared the survival of wild-type or Atg5�/� cells

(Kuma et al., 2004) following proteasome inhibition. We found


A B

C D

E

Figure 4. Amino Acid Supplementation Suppresses Autophagy Resulting from Proteasome Inhibition

(A) Confocal micrographs of HeLa cells stably expressing GFP-LC3 (Thurston et al., 2009) either untreated or following 10 mMMG-132 treatment, with or without

1 mM Cys, where indicated. Nuclei were stained with H33258.

(B) Quantification of GFP-LC3 dots in at least 100 cells exposed to the indicated treatment. Data are means ± SEM. ***p % 0.0001.

(C) LC3 and tubulin immunoblots of lysates from cells treated with 100 nMBafilomycin A1 (Baf A1) for 15 hr, or 10 mMMG-132 alone or together with 1mMCys for

the indicated time.

Molecular Cell


248 Molecular Cell 48, 242–253, October 26, 2012 ª2012 Elsevier Inc.

Molecular Cell


that Atg5�/� cells were significantly more sensitive to MG-132

treatment and this was manifest already following 1 hr of protea-

some inhibition (Figure 4E).

Themammalian target of rapamycin (mTOR) signalingpathway

senses and responds to amino acid availability to modulate

protein synthesis via the phosphorylation of substrates such as

S6 kinase 1 (S6K1) (Ma and Blenis, 2009) (Figure 5A). To evaluate

whether autophagy contributed to replenish the pool of intracel-

lular amino acids upon proteasome inhibition, we measured the

levels of phosphorylation of threonine 389 of the mTORC1

substrate S6K1 (Ma and Blenis, 2009). In agreement with the

decrease in critical amino acids following proteasome inhibition,

S6K1 phosphorylation decreased 1 hr after MG-132 addition

(Figure 5B). Four hours following MG-132 addition, S6K1 phos-

phorylation increased (Figure 5B). This increase was abrogated

by Bafilomycin A1, indicating that it was dependent on autopha-

gic degradation (Figure 5B). Amino acid sensing by mTOR is

mediated by the Rag GTPases and dominant negative mutants

of the Rag GTPases abrogate mTOR stimulation by amino acids

(Sancak et al., 2008). Strikingly, overexpression of dominant

negative mutants of the Rag GTPases that prevent amino acid

sensing by mTORC1 (Figure 5A) also abolished the increased

phosphorylation of S6K1 that followed proteasome inhibition

(Figure 5C). These results reveal that amino acid scarcity is the

signal that induces autophagy when the proteasome is inhibited,

in an attempt to rescue amino acid homeostasis.

This model predicts that blocking amino acid consumption by

inhibiting protein synthesis should recapitulate the rescuing

effects of amino acid supplementation in proteasome inhibited

cells. Indeed, we found that addition of cycloheximide together

with MG-132 prevented the decreased S6K1 phosphorylation

resulting from treatment with MG-132 for 1 hr (Figure 5D). Con-

sistently, the decrease in cysteine and asparagine/aspartate re-

sulting from proteasome inhibition was markedly attenuated in

cells cotreated with cycloheximide (Figure 5E). Strikingly, cyclo-

heximide protected cells from the lethality resulting from protea-

some inhibition (Figure 5F). The rescuing effect of cycloheximide

in proteasome-inhibited cells was selective because cyclohexi-

mide increased the lethality of cells exposed to UV irradiation

(data not shown). Thus, inhibition of protein synthesis markedly

attenuates the depletion of critical amino acid following protea-

some inhibition as well as the lethality resulting from proteasome

inhibition.

An Amino Acid Imbalance Underlies the LethalityResulting from Proteasome Inhibition in Drosophila

We next assessed whether proteasome inhibition had detri-

mental consequences on amino acid homeostasis in an organ-

ismal context. We exposed Drosophila to proteasome inhibitors

and monitored amino acid levels. Bortezomib significantly

reduced the levels of asparagine/aspartate, threonine/serine,

glutamine/glutamate, phenylalanine, and proline in Drosophila

(D) Quantification of the LC3-II/tubulin ratio in three independent experiments. V

(E) Viability of wild-type and Atg5�/� mouse embryonic fibroblasts, assessed

treatment with 10 mMMG-132 for the indicated time and a washout in regular med

not significant.

See also Figure S4.

Mol

(Figure 6A). Bortezomib also increased the levels of glycine (Fig-

ure 6A), similar to prolonged starvation in humans (Felig et al.,

1969). As expected (Vernace et al., 2007), exposure of flies to

Bortezomib was lethal (Figure 6B). Amino acid supplementation

markedly increased survival of proteasome-inhibited flies (Fig-

ure 6B), without detectable changes in the levels of proteasome

substrates (Figure 6C). Thus, proteasome inhibition caused an

intolerable amino acid imbalance in flies and the resulting

lethality was rescued upon supplementation with amino acids.

DISCUSSION

Here, we show that the detrimental consequences of protea-

some inhibition are largely abrogated by supplementation with

amino acids in yeast,mammalian cells, andDrosophila (Figure 7).

Strikingly, the rescuing effect of amino acids occurs without

reducing the accumulation of proteasome substrates. These

findings reveal that cells can survive with the protein waste

they accumulate when the proteasome is inhibited but the result-

ing amino acid deficiency is lethal. Thus, the proteasome

substrates accumulating upon proteasome inhibition appear

detrimental to cells as they sequester a pool of critical amino

acids that would otherwise be recycled.

Although abrogating death of proteasome-inhibited cells,

amino acid supplementation does not abrogate the requirement

for proteasomal degradation for regulatory functions such as cell

division. The amino acid scarcity resulting from proteasome

inhibition induces the classical set of responses to amino acid

starvation, in an vain attempt to sustain amino acid homeostasis.

Although it had been previously reported that proteasome inhibi-

tion, like many different stresses, induces eIF2a phosphorylation

and that the resulting translation attenuation is abrogated by

genetic impairment of eIF2a phosphorylation (Jiang and Wek,

2005), the underlyingmechanismwas unknown.We have shown

here that the amino acid decrease following proteasome

inhibition is the signal that activates GCN2 and induces eIF2a

phosphorylation, leading to attenuation of protein synthesis (Fig-

ure 3). Cysteine is the least abundant cellular amino acid and we

found that cysteine scarcity following proteasome inhibition

impairs protein synthesis. Upon persistent inhibition of the pro-

teasome and the subsequent amino acid insufficiency, the ISR

is chronically activated, thereby leading to the expression of

the pro-apoptotic protein CHOP (Figure 3B). Supplementation

with a critical amino acid, depleted upon proteasome inhibition,

virtually suppresses ISR signaling and cell death (Figure 3B),

similar to genetic ablation of CHOP (Figure 3C) or impairment

of eIF2a phosphorylation, which reduces death following protea-

some inhibition (Jiang and Wek, 2005).

The mechanisms underlying the crosstalk between autopha-

gic and proteasomal degradation have remained elusive. In

this study, we provide the missing link between autophagy

induction and proteasome inhibition. We show that the signal

alues were normalized to untreated cells. Data are means ± SEM. *p % 0.05.

by the reduction of WST-8 into formazan, after 2 days of growth, following

ium. Data are means ± SD (n = 4), normalized to cell number. **p% 0.001. n.s.,


A

C

MG-132 0 1 2 3 4 8 0 1 2 3 4 8 (h) Mock transfected RagB-DN transfected

S6K1

FLAG

P -T389-S6K1

Baf A1

MG-132 0 1 2 3 4 8 0 1 2 3 4 8 (h)

P -T389-S6K1

S6K1

B

Amino acids

RagA/B

RagC/D

GTP

GDP

mTOR

RagA/B

RagC/D

GDP

GTP

P -T389-S6K1

1 h

MG

-132

D

S6K1

P -T389-S6K1

E

% s

urvi

val

CHX - +

F

MG-132 10 µM

Cys AsxUT 100 100MG-132 73.3 ±1.9 64 ±2.7MG-132 + CHX 88.6 ±1.6 97.7 ±3

1 h

MG

-132

+

CH

X

***

Figure 5. Autophagy and Rag GTPases-Dependent Amino Acid

Signaling to mTOR upon Proteasome Inhibition

(A) Model for amino acid signaling to mTOR by the Rag GTPases (RagA/BGTP-

RagC/DGDP). Amino acid sensing by the Rag GTPases is blocked by dominant

negative mutants (RagBGDP-RagCGTP) (Sancak et al., 2008).

Molecular Cell



responsible for autophagy induction upon proteasome inhibition

is the resulting amino acid scarcity, the canonical inducer of

autophagy. Autophagy aims at protecting cells from the delete-

rious consequences of proteasome inhibition, as cells lacking

Atg5, a key autophagy component are more sensitive to protea-

some inhibition. Both the ISR and autophagy are involved as

early as 1 hr following proteasome inhibition to adapt cells to

the resulting changes. Our work shows that autophagy and

proteasomal degradation act in a concerted manner to adjust

the supply of amino acids to the cellular needs. Following pro-

teasome inhibition, autophagy attempts to restore amino acid

homeostasis, as manifested by an increased S6K1 phosphoryla-

tion via the canonical, Rag GTPases-dependent amino acid

signaling pathway to mTOR. The findings reported here reveal

how changes in amino acid homeostasis are integrated with

protein metabolism and cell death (Figure 7).

We found here that the deleterious consequences of protea-

some inhibition are rescued upon amino acid supplementation

in yeast, mammalian cells, andDrosophila. These findings estab-

lish the importance of proteasomal degradation in recycling

amino acids for normal cell and organismal function. There is

a qualitative difference in the required amino acids in the three

different systems that is not surprising as yeast, mammalian

cells, andDrosophila have different properties and requirements.

Yeast cells required a broad range of amino acids to survive pro-

teasome inhibition, perhaps because they have higher metabolic

needs, as they divide more rapidly than mammalian cells. Upon

proteasome inhibition, mammalian cells had a prominent need

for cysteine and asparagine and we show that the decrease in

cysteine and asparagine/aspartate were largely prevented by

blocking protein synthesis in proteasome-inhibited cells (Fig-

ure 5E). Notably, enzymes required for the metabolism of

cysteine and asparagine, two conditionally essential amino

acids, are under the control of the ISR, thus underscoring an

evolutionary pressure to maintain the levels of asparagine and

cysteine in the cell. In addition, the importance of asparagine

starvation has been previously recognized and is the basis for

the use of L-asparaginase for the treatment of acute lympho-

blastic leukemia (Muller and Boos, 1998).

The proteasome inhibitor Bortezomib is used in cancer

therapy but it is limited to the treatment of multiple myeloma

(B) Immunoblots of lysates from 293T cells exposed to 10 mM MG-132 treat-

ment revealed with phospho-T398-S6K1 or S6K1 antibodies. Cells were either

left untreated or treated with 100 nM Baf A1 for the indicated time.

(C) Immunoblots with phospho-T398-S6K1 or S6K1 antibodies of lysates of

cells mock transfected or transfected with dominant negative forms of RagB

(RagB-DN, FLAG-tagged).

(D) Immunoblots with phospho-T398-S6K1 or S6K1 antibodies of lysates of

293T cells either left untreated or treated with 10 mM MG-132 with or without

100 mM cycloheximide (CHX) for 1 hr.

(E) Changes in the content of cellular free cysteine (Cys) or asparagine/

aspartate (Asx) in cells treated with 10 mM MG-132 with or without 50 mM

cycloheximide (CHX) for 4 hr.

(F) Viability of NIH 3T3 cells, assessed by the reduction of WST-8 into

formazan, after 2 days of growth, following treatment with 10 mMMG-132 with

or without 50 mM cycloheximide (CHX) for 8 hr. Data are means ± SD (n = 4),

normalized to cell number. ***p % 0.0001. Representative results of at least

three independent experiments are shown.

.

A

Am

ino

acid

(nm

ol)

**

**

***

***

B

0

20

40

60

80

100

0 1 2 3 4

Untreatedno AA+ AA

Bortezomib treated

***

**

**

C

Ponceau

Unt

reat

ed

-

Bortezomib

+ A

A

Ubn

***

Sur

viva

l (%

)

Days

**

Figure 6. Amino Acid Imbalance Mediates

the Lethality Resulting from Proteasome

Inhibition in Drosophila

(A) Changes in the content of free amino acids

in Drosophila treated with or without 50 mM

Bortezomib for 3 days. Asx, asparagine/aspartate;

Glx, glutamine/glutamate. Data are means ± SEM

(30 flies per conditions, from six independent

experiments). **p % 0.005; ***p % 0.001.

(B) Survival of Drosophila either untreated or

treated with 50 mM Bortezomib, with or without

additional amino acids (AA), at the concentration

used in Grandison et al. (2009). Data are means ±

SEM (400 flies per condition, from four indepen-

dent experiments). **p % 0.005; ***p % 0.001.

(C) Polyubiquitin (Ubn) immunoblots from lysates

of flies treated with 50 mM Bortezomib and

additional amino acids, where indicated. Ponceau

staining of the membrane. Representative results

of at least three independent experiments are

shown.

Molecular Cell


and restricted by adverse effects, such as peripheral neuropathy

(Richardson et al., 2003). So far, the mechanisms by which

proteasome inhibitors kill cells have remained elusive. Failure

of amino acid homeostasis not only explains the lethality result-

ing from proteasome inhibition but also provides a foundation for

the development of novel cancer therapies.

EXPERIMENTAL PROCEDURES

Mammalian Cell Culture

NIH 3T3, 293T (ATCC), 293T cells stably expressing ZsGreen-ODC

(ZsProsensor-1 cells, Clontech), and HeLa cells were maintained at 37�C in

DMEM supplemented with 10% fetal bovine serum (FBS), penicillin, and

streptomycin. MEFs cells were cultured in DMEM supplemented with

Molecular Cell 48, 242–253,

penicillin, streptomycin, glutamine, 13 nonessen-

tial amino acids (Sigma), sodium pyruvate, and

10% FBS.

Assessment of Cell Viability

Cells were plated (day 0) in DMEM supplemented

with 10% FBS bovine serum in 24-well plates

(Nunc), at a density of 8,000 cells/ml. The next

day (day 1), media were replaced with DMEM

supplemented with 0.5% FBS. On day 2, cells

were treated with proteasome inhibitors for the

indicated period of time. Media containing pro-

teasome inhibitors were replaced with DMEM

supplemented with 0.5% FBS and the indicated

additives. Four wells per condition were used.

On day 3, media were replaced with DMEM

supplemented with 10% FBS. Cells were kept

for 2–3 days as indicated. Cell viability was

assessed by measuring the reduction of WST-8

[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-

(2,4-disulfophenyl)-2H-tetrazolium] into formazan

using Cell Viability Counting Kit-8 (Dojindo) ac-

cording to the supplier’s recommendation.

Flow Cytometry and Confocal Microscopy

Flow cytometry analyses were performed on

a FACSCalibur flow cytometer (BD Biosciences)

and analyzed with FlowJo and confocal microscopy was carried out as

described in Munch et al. (2011).

Immunoblot Analyses and Assessment of Translation Rates

Cell lysis, immunoblots, and measurement of translation rates were performed

as described in Tsaytler et al. (2011). Note that cells were not starved in methi-

onine-free media before labeling with 35S-methionine for monitoring protein

synthesis.

Yeast Strains, Media, and Culture

Cim3-1 or pre1-1 and their respective isogenic wild-type strains (Ghislain

et al., 1993; Heinemeyer et al., 1991) and ndc80-1 (Wigge et al., 1998) were

grown in YPD medium using standard techniques, as in Dehay and Bertolotti

(2006).

October 26, 2012 ª2012 Elsevier Inc. 251

Figure 7. Proteasome Inhibition Causes an Intolerable Amino Acid Imbalance and the Resulting Lethality Is Rescued upon Supplementation

with Amino Acids

The amino acid shortage resulting from proteasome inhibition is the signal that induces eIF2a phosphorylation and autophagy, in a vain attempt to replenish

to pool of amino acid and sustain vital protein synthesis rates. In yeast, mammalian cells, and Drosophila, the lethality resulting from proteasome inhibition is

rescued upon amino acid supplementation. This rescuing effect occurs without a detectable decrease in the levels of proteasome substrates in proteasome-

inhibited cells.

Molecular Cell


Amino Acid Analyses

Amino acid analysis from yeast cells and mammalian cells were performed

as described, respectively (Onodera and Ohsumi, 2005; Vabulas and Hartl,

2005).

Flies

Wild-type Oregon R flies were reared on standard food. Amino acids were

extracted in 600 ml methanol from 5 frozen female flies per condition, as in

Hayward et al. (1993). The suspension was centrifuged and the supernatant

collected, vacuum-dried, and analyzed. Some amino acids were below detec-

tion levels in such analyses. For survival experiments, 20 flies, both females

and males, were kept in vials lined with 3 mm filter paper and fed a solution

of 5% sucrose with either vehicle (0.5% DMSO, control) or with Bortezomib

(50 mM in DMSO), for the indicated time, as in Vernace et al. (2007). Under

the same conditions MG-132 (50 mM in DMSO) was not toxic. In Figure 6B,

1.5 ml of 5% sucrose with or without amino acids at the concentration used

in Grandison et al. (2009) was added together with the proteasome inhibitor.

Proteins were extracted from five frozen female flies per conditions in 250 ml

boiling Laemmli buffer supplemented with 10 mM NEM and 50 mM DTT.

Statistical Analysis

Data are presented as means and SD or SEM, as indicated. Where indicated,

two groups were compared with a two-tailed nonpaired Student’s t test.

SUPPLEMENTAL INFORMATION

Supplemental Information includes four figures and Supplemental Experi-

mental Procedures and can be found with this article online at http://dx.doi.

org/10.1016/j.molcel.2012.08.003.

ACKNOWLEDGMENTS

We are grateful to D. Owen for amino acid analysis; C. Mann, D.H. Wolf, J. Kil-

martin, M. Lewis, N. Mizushima, B. McGrath, D. Cavener, H. Harding, D. Ron,

W. Greene, M. Narita, and F. Randow for reagents; M. Freeman, S. Aldaz


Casanova, and M. Zettl for help and advice on the work with Drosophila;

Z. Zhong for help with the revision; G. Lingley and P. Margiotta for help with

figures and illustrations; and M.M. Babu, M. Goedert, M.H. Hastings, D.

Komander, and S. Munro for discussions. This work was supported by the

UK Medical Research Council.

Received: February 2, 2012

Revised: June 4, 2012

Accepted: August 3, 2012

Published online: September 6, 2012

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