Bioscience Reports i, 35-44 (1981)
Printed in Great Britain
35
The regulation of protein synthesis in mammalian
c e l l s by a m i n o acid supply
Sara A. AUSTIN and Michael 3. CLEMENS
Department of Biochemistry, St. George's Hospital
Medical School, Cranmer Terrace, London SWI7 ORE, U.K.
(Received 18 December 1980)
This review is concerned with the translational control of protein
synthesis exhibited by a variety of mammalian cell types in r e s p o n s e
to c h a n g i n g n u t r i t i o n a l c o n d i t i o n s .
The m o l e c u l a r basis of this
phenomenon is now partially understood, and it has provided a number
of insights into c y t o p l a s m i c control mechanisms in eukaryotes. We
describe here the current state of knowledge about these mechanisms
and~ in particulaG the role played by the polypeptide chain initiation
factor eIF-2.
We also b r i e f l y c o n s i d e r how cells may r e c o g n i z e
c h a n g e s in the a v a i l a b i l i t y of nutrients9 especially essential amino
acids~ and how they adapt to short-term or long-term deprivation of
these compounds which are needed for the accumulation o5 protein and
for growth.
The Evidence for Translational Control
There is much e v i d e n c e to i n d i c a t e t h a t p r o t e i n s y n t h e s i s in
mammalian cells in tissue culture is closely regulated by the supply of
essential nutrients (van Venrooij e t a l . ,
1970; Vaughan e t a l . ,
1971; Lee e t a l . ,
1971; van Venrooij e t a l . ,
1972; Henshaw9
1980).
In such ceil% deprivation of amino acid% glucos% or serum
r e s u l t s in a reduction in the rate of protein synthesis. This occurs
immediately in cells deprived of essential amino acids (van Venrooij
et al.,
1970; van Venrooij e t a l . ,
1972; Vaughan e t a l . ,
1971)
and within l h in ceils deprived of glucose (van Venrooij e t a l . ,
1972). The e f f e c t of serum starvation becomes apparent only at later
times (Kaminska% 1972).
Refeeding amino acid-deprived cells with
t h e missing amino acid r e s u l t s in a rapid recovery of the rate of
protein synthesis (van Venrooij e t a l . ,
1970; Vaughan e t a l . ,
1971;
van Venrooij e t a l . ,
I972; Sonenshein & Brawerman, 1977).
These
events occur even in the p r e s e n c e of s u f f i c i e n t a c t i n o m y c i n D to
inhibit all new RNA synthesis (van Venrooij e t a l . ,
1970; Vaughan
et al., 1971; Lee et al., 1971; Sonenshein & Brawerman~
1977) 7
i n d i c a t i n g t h a t t r a n s l a t i o n a l r a t h e r than t r a n s c r i p t i o n a l c o n t r o l
mechanisms are operative. The rapidity of the response of cells to
refeeding with the missing amino acid is in itself further evidence for
translational control. Fig. 1 i l l u s t r a t e s this point.
The r a t e s of
incorporation
of [ 1 4 C ] l e u c i n e
into protein are shown for
l y s i n e - d e p r i v e d Ehrlich a s c i t e s t u m o u r ceils and for s u c h c e i l s
i m m e d i a t e l y a f t e r r e f e e d i n g with the missing amino acid. Lysine
deprivation results in a 60~ reduction in the rate of protein synthesis
(Pain e t a l . ,
1980), but on refeeding with lysine the rate recovers
within 1 to 2 min. As this is approximately the length of time a cell
takes to make a complete polypeptide chain, the response to the refed
amino acid is virtually instantaneous.
9
The
Biochemical
Society
36
S.A. AUSTIN & M.3. CLEMENS
3
2
6"
Q
i
E
O
0
I
I
5
I0
I
15
Time (rain)
Fig. i.
Kinetics of recovery of cells from amino
acid starvation.
Ehrlich ascites tumour cells were
incubated in lysine-free culture medium for 30 min at
37~
Lysine was then added to one b a t c h a n d
protein synthesis measured from the incorporation of
[14C]leucine
(0.4 mM,
1.25
Ci/mol)
into
acid-insoluble
m a t e r i a l (Pain et a 2 . , 1980).
Incorporation into 0.5-ml aliquots of cell suspension
(1.15 x 106 cells) is shown for each time.
(Q),
starved cells; (O)~ refed cells.
The Mechanism of R e g u l a t i o n
A s s o c i a t e d with the reduction in protein synthetic rate in amino
a c i d - s t a r v e d c e l l s t h e r e is m a r k e d p o l y s o m e d i s a g g r e g a t i o n a n d
accumulation of inactive 80-S ribosomes (van Venrooij e e a 2 . ,
1970;
Vaughan e t a 2 . ,
1971; Lee e t a 2 . ,
1971; van Venrooij e t a 2 . ,
i972).
This indicates a blockage in ribosome a t t a c h m e n t to mRNA
during polypeptide-chain initiation. There may also be smaller e f f e c t s
on the rate of polypeptide-chain elongation (Vaughan e t a l . ,
1971;
van Venrooij e t a l . ,
1972), but as initiation is the rate-limiting step
f o r o v e r a l l t r a n s l a t i o n , these do not become apparent under normal
circumstances.
In Ehrlich ceils deprived of lysine there is a substantial reduction
in the number of [tt0-S Met t R N A f ] initiation complexes formed on
native small ribosomal subunits (Pain & Henshaw, 1975), indicating that
in starved cells initiation is controlled at this point.
The e f f e c t s of
such starvation are preserved in c e l l - f r e e e x t r a c t s prepared from fed
and l y s i n e - d e p r i v e d c e i l s ( P a i n e e a l . ,
1980).
In
vitro
the
decrease in the level of 40-S initiation complexes can be reversed by
the addition of the polypeptide-chain initiation factor eIF-2 (Pain e t
REGULATION OF PROTEIN SYNTHESIS
37
al.,
1980), as illustrated in Fig. 2. Interestingly, refeeding with the
missing amino acid has no r e s t o r a t i v e e f f e c t i n v i t r o
(Pain et
al.,
1980).
It appears that the depression in the rate oI initiation
of p o l y p e p t i d e chains in a m i n o a c i d - d e p r i v e d ceils is c a u s e d by
inactivation of elF-2, a control mechanism which is also seen in the
reticulocyte system in its response to haem d e f i c i e n c y .
It is well
documented that protein synthesis in reticulocytes, and in lysates from
these cells, is dependent upon the supply o5 haem (Austin & Clemen%
r
Fed
I
,,
0.6
0.3
8
~
o
I
I Fed+
I eIF-2
,
!
~
I I eIF-2
0.6 ~
0.3
60S
I0
15
I0
Fraction Number
15
Fig. 2. Effect of initiation factor elF-2 on the level of
40-S initiation complexes in e x t r a c t s from fed and
lysine-deprived Ehrlich ascites tumour cells.
Extracts
from fed cells and from cells starved of lysine for 30 min
were incubated with
[35S]methionine (160 pCi/ml) for 2
min in the presence and in the absence of 44 pg/ml elF-2.
The samples were fractionated by sucrose-density-gradient
centrifugation (sedimentation was from right to left), and
the r a d i o a c t i v i t y
in 40-S initiation complexes was
determined as described by Pain et al., (1980).
optical density at 260 nm; Q - - - Q ,
radioactivity (counts
per minute per fraction). The positions of 40-$9 60-S~ and
80-S ribosomal particles are indicated~ and the radioactive
methionine associated w i t h 4 0 - S - s u b u n i t
initiation
complexes is shown by the shaded areas.
38
S.A. AUSTIN & M.3. CLEMENS
1980b; C l e m e n s , 1980; Ochoa & de Haro, 1979).
In the absence of
haem, synthesis stops, polysomes disaggregate, and the l e v e l of 40-S
initiation complexes declines (Legon e e a 2 . , 1973). Associated with
this is the activation of a protein kinase, named the haem-controlled
repressor (HCR) or haem-regulated inhibitor (HRI), which phosphorylares the small subunit ol initiation factor eIF-2 (Ranu & London, 1976;
Farrell ee a2.,
1977).
P h o s p h o r y l a t i o n of this f a c t o r has been
closely correlated with its inactivation, although other interactions are
now known to be necessary since phosphorylated eIF-2 itself is still
active in f r a c t i o n a t e d c e l l - f r e e s y s t e m s ( F a r r e l l e e a 2 . ,
1977;
Trachsel & Staehelin, 1978).
In c e l l - f r e e e x t r a c t s f r o m fed and lysine-deprived Ehrlich cells,
eIF-2 is also phosphorylated, but in this case we have been unable to
d e t e c t any i n c r e a s e in p h o s p h o r y l a t i o n in e x t r a c t s prepared from
starved cells (S.A. Austin & M.~. Clemens, manuscript in preparation).
It is known t h a t t h e s e c e i l s possess a p r o t e i n kinase which can
phosphorylate the small subunit of eIF-2, as well as a p h o s p h a t a s e
which d e p h o s p h o r y l a t e s it (S.T. Wong and E.C. Henshaw, personal
communication), but at p r e s e n t t h e role of t h e s e e n z y m e s in t h e
starvation-induced inactivation of eIF-2 remains unclear. It is possible
that, as in the reticulocyte, phosphorylation of eIF-2 is a n e c e s s a r y
prerequisite for its inactivation, but some further interactions may also
be involved.
Studies of the subcellular distribution of eIF-2 in Ehrlich cells have
revealed that there is less active factor on starved cell ribosomes than
on fed cell ribosomes, at least in a form which can be washed off
with 0.5 M KC1 (Clemens & Bloyce, 1980).
This correlates with the
inability of starved ceils to form 40-S initiation complexes efficiently.
However, the interpretation of such data is complicated by t h e f a c t
that when e x t r a c t s are prepared from Ehrlich ceils a high proportion
of the total recoverable elF-2 activity sediments with the nuclear and
m i t o c h o n d r i a l p e l l e t , which is usually d i s c a r d e d (Clemens & Pain,
1981). It is not yet known whether this eIF-2 is functionally similar
to that normally found associated with ribosomes.
Because of the obvious mechanistic similarities between the control
of p r o t e i n s y n t h e s i s in E h r l i c h cells by amino acid supply and the
e f f e c t s of haem on t r a n s l a t i o n in r e t i c u l o c y t e s , we have r e c e n t l y
e x a m i n e d t h e e f f e c t s of adding haem to Ehrlich-cell-free e x t r a c t s
(S.A. Austin & M.3. Clemens, manuscript in preparation).
The results
of this study have shown that in e x t r a c t s from fed ceils haem quite
markedly stimulates protein synthesis and the level of 40-S initiation
complexes. In e x t r a c t s from starved cells, this stimulation appears to
be blocked by the action of an inhibitor.
Preliminary evidence also
suggests that this inhibitor can partially abolish the haem stimulation
of initiation when preparations from fed and starved cells are mixed
t o g e t h e r (S.A. Austin & M.3. C l e m e n s , manuscript in preparation).
H o w e v e r , t h e p r e s e n c e of t h e i n h i b i t o r is n o t a l w a y s r e a d i l y
d e m o n s t r a t e d in o t h e r types of assay.
For example, e x t r a c t s from
both fed and s t a r v e d c e i l s , when added to h a e m - s u p p l e m e n t e d
r e t i c u l o c y t e l y s a t e , can i n h i b i t i n i t i a t i o n at the level of the tt0-S
initiation complex. A possible model for the relationship between this
inhibition, the starvation-induced inhibition, and the phosphorylation of
eIF-2, is summarized in Fig. 3. By analogy with t h e r e g u l a t i o n of
REGULATION
OF
PROTEIN
SYNTHESIS
39
elF-2
regulation
by haem
5
Q
ffl
40S. Met-tRNA 9GTP]
f
eIF-2@
regulation by amino
acid starvation
1
Fig. 3.
A m o d e l for the regulation of elF-2dependent polypeptide chain initiation by haem and by
amino acid starvation.
Initiation factor elF-2 is
depicted as existing in both unphosphorylated and
phosphorylated states, distribution between which is
controlled by the relative activites of a protein
kinase and a phosphatase.
It is proposed that haem
regulates the activity of the protein kinase, as in
reticulocytes.
Both forms of the factor may be
active in initiation, but it is proposed that elF-2 P
can interact with inhibitory components I and X,
which impair its availability for protein synthesis.
The complex with I may exist in extracts from fed
cells and would be dissociable.
X is shown as being
activated by amino acid starvation and forming a
non-dissociable complex with elF-2 P.
For more
details, and the evidence supporting this model, see
the text.
eIF-2 a c t i v i t y in the r e t i c u l o c y t e s y s t e m ( F a r r e l l e e a 2 . ,
1977), we
propose t h a t the a d d i t i o n of h a e m to e x t r a c t s
from Ehrlich cells
inhibits a p r o t e i n kinase with s p e c i f i c i t y for eIF-2, thus i n c r e a s i n g the
r a t i o of u n p h o s p h o r y l a t e d to p h o s p h o r y l a t e d f a c t o r .
This in itself m a y
not s t i m u l a t e initiation, since both f o r m s of eIF-2 a r e a c t i v e ( a t l e a s t
in vitro)
(Farrell ee a2.,
1977; T r a c h s e l & S t a e h e l i n , 1 9 7 8 ) .
H o w e v e r , we p r o p o s e t h a t eIF-2 P binds to inhibitors, d e p i c t e d as I
and X in Fig. 3, which i n a c t i v a t e it. Inhibitor I is b e l i e v e d to exist in
b o t h f e d and a m i n o a c i d - s t a r v e d
c e l l s , a n d c o m p l e x e s of it with
eIF-2 P a r e d e p i c t e d as being in equilibrium with the f r e e p h o s p h o r y l a t e d initiation f a c t o r .
Thus when the level of the l a t t e r is l o w e r e d
by the addition of h a e m , dissociation of the c o m p l e x with I would be
f a v o u r e d a n d , in e x t r a c t s
f r o m f e d c e i l s , t h i s w o u l d l e a d to an
i n c r e a s e in the level of a c t i v e eIF-2.
However, we postulate that
s t a r v a t i o n a c t i v a t e s or c a u s e s the a p p e a r a n c e of the inhibitor X, which
does not d i s s o c i a t e f r o m eIF-2 P on the addition of h a e m .
Thus no
f u r t h e r a c t i v e eIF-2 is r e l e a s e d for initiation and t h e r e is no e f f e c t of
#0
S.A. AUSTIN & M.J. CLEMENS
haem.
As y e t we do not know the n a t u r e of I or X. They could be
r e l a t e d , but need not be so.
A l t e r n a t i v e l y , I or X m a y r e p r e s e n t
modifications
to e I F - 2 , r a t h e r t h a n d i s c r e t e m o l e c u l a r e n t i t i e s .
F u r t h e r a s p e c t s of t h i s m o d e l a r e c u r r e n t l y b e i n g s t u d i e d in o u r
laboratory.
C e l l u l a r R e s p o n s e s to Amino Acid S t a r v a t i o n
It is of interest to consider the possible ways in which the lack of
one essential amino acid in the extracellular medium results in an
inhibition of polypeptide chain i n i t i a t i o n inside the cell.
In other
words, what are the signals which the cell recognizes? One possible
candidate for such a signal could be a change in the levels of
endogenous guanine or adenine nucleotides in starved cells.
It has
been shown by others that intracellular pools of ATP and GTP decline
in size f o l l o w i n g starvation for amino acids (van Venrooij e t a l . ,
1972; Grummt &Grummt, 1978).
However, in Ehriich cells under the conditions we have used, the
levels of ATP and GTP and their corresponding di- and monophosphates
(measured by high-performance liquid chromatography) did not change
significantly during amino acid s t a r v a t i o n , at least <at early times
(S.A. A u s t i n & D. Perrett, unpublished observations). The published
studies (van Venrooij e t a l . ,
i972; Grummt & Grummt, I978) have
only looked at later times, i.e., l - 3 h after starvation was started,
when a whole range of cellular functions in addition to protein
synthesis may well have been affected (see below).
Within the first
30 min of a m i n o acid d e p r i v a t i o n , when p r o t e i n synthesis is rapidly and
r e v e r s i b l y inhibited, a change in nucleOtide levels s u f f i c i e n t to a c c o u n t
for this response a p p e a r s i m p r o b a b l e .
It h a s f r e q u e n t l y b e e n s u g g e s t e d t h a t it is the a c c u m u l a t i o n of
u n c h a r g e d t R N A in a m i n o a c i d - s t a r v e d cells t h a t c a u s e s the inhibition
of p o l y p e p t i d e - c h a i n
i n i t i a t i o n (Vaughan & Hansen, 1973; G r u m m t &
G r u m m t , 1978).
We now no longer believe this to be t h e c a s e , a t
l e a s t as far as the #0-S initiation c o m p l e x is c o n c e r n e d . S e v e r a l lines
of e v i d e n c e a r g u e a g a i n s t u n c h a r g e d t R N A being involved at this level.
Firstly, if Ehrlich ceils a r e t r e a t e d with a low dose of c y c l o h e x i m i d e
and s i m u l t a n e o u s l y s t a r v e d for lysine, p o l y p e p t i d e - c h a i n e l o n g a t i o n is
inhibited m o r e than initiation, and p o l y r i b o s o m e s a r e m a i n t a i n e d in an
undegraded state.
Under t h e s e c o n d i t i o n s all t R N A s will be f u l l y
c h a r g e d , as t h e a m i n o acids a r e being utilized for p r o t e i n synthesis
only v e r y slowly.
S u f f i c i e n t lysine is p r o v i d e d f r o m p r o t e i n d e g r a dation to m e e t the r e q u i r e m e n t for p r o t e i n synthesis.
H o w e v e r , the
r e d u c t i o n in the level of #0-S initiation c o m p l e x e s is still o b s e r v e d in
e x t r a c t s f r o m such s t a r v e d cells (Austin & C l e m e n s , 1 9 8 0 a ) .
The d a t a
a r e s u m m a r i z e d in T a b l e 1.
A s e c o n d p i e c e of e v i d e n c e c o n c e r n s the e f f e c t s of d e a c y l a t e d
t R N A which has been oxidized at t h e 3' end with p e r i o d a t e to p r e v e n t
recharging with amino acids.
A d d i t i o n of such t R N A to c e l l - f r e e
e x t r a c t s f r o m fed cells has v e r y little e f f e c t on t h e l e v e l of # 0 - S
initiation
c o m p l e x e s , a l t h o u g h i t is s t r o n g l y i n h i b i t o r y t o 8 0 - S
c o m p l e x e s (Austin e t a 2 . ,
1980).
T h i r d l y , we h a v e f o u n d t h a t
extracts
f r o m b o t h fed and s t a r v e d cells h a v e similar c a p a c i t i e s to
c h a r g e t R N A with a m i n o acids, including the one of which the ceils
R E G U L A T I O N OF
PROTEIN
SYNTHESIS
#I
Table I, 40-S initiation complex formation in cell-free extracts
from fed and lysine-deprived Ehrlich ascites tumour cells with
or without exposure to a low concentration of cycloheximide
Type of extract
~5S]Methionine bound to
40-S initiation complexes
(pmol/A260 unit of extract)
Fed
0.223
Starved
0.049
Fed + cycloheximide
0.142
Starved + cycloheximide
0.027
Two pairs of cell-free extracts from fed and lysine-deprived
cells were prepared as described in Pain et al. (1980).
One pair of fed and starved extracts was p r e p a r e d by
incubating the cells with 0.05 pg/ml cycloheximide (to
partially inhibit elongation of polypeptide chains) during
the 30-min starvation procedure.
Cell-free extracts from
these cells were then prepared as usual.
40-S initiation
c o m p l e x f o r m a t i o n was m e a s u r e d in these extracts as
described in Pain et a2. (1980), and the amount of [35S]
methionine bound to 40-S ribosomal subunits expressed as
pmol/A260 unit of extract.
have been d e p r i v e d .
H o w e v e r , as s t a t e d earlier, under conditions
where extracts from lysine-deprived cells are supplemented with lysine
to equal or exceed the amount found in extracts from led cells, the
level of 40-S initiation complexes is still reduced (Pain e t a l . ,
[9g0;
Austin & Clemens) 1980a).
Taken together these various observations
suggest that, in mammalian cells, the control oi protein synthesis by
amino acid supply does not depend critically on the accumulation of
deacylated t R N A , at ]east with respect to the 40-5 initiation complex.
This lack of dependence is in contrast to the stringent response to
amino acid starvation
in b a c t e r i a ,
w h i c h is a c t i v a t e d
by
ribosome-bound deacylated t R N A (Haseltine & Block, 1973).
The evidence against the involvement os either changes in nucleotide pool sizes or variations in Jevels of charging of t R N A in eliciting
the effects of amino acid starvation on p o l y p e p t i d e - c h a i n i n i t i a t i o n
therelore leaves unresolved the probJem os how cells recognize their
altered nutritional state.
Whatever mechanism is involved works very
rapidly in intact cells (Fig I . ) .
This instant response, contrasted w i t h
the complete refractoriness of ce11-free systems, suggests a role for
some c e l l u l a r f r a c t i o n which is discarded during the preparation of
extracts from cells. The plasma membrane could be one candidate for
such a fraction.
Alternatively, a highly l a b i l e ' a m i n o acid receptor' or
similar intracellular component may be involved, but as yet we have
no idea what this could be or how it might work.
#2
Long-term Adaptations
S.A. AUSTIN & M.3. CLEMENS
to
Nutritional Deprivation
The m a j o r i t y of the changes described above are very r a p i d
responses of cells in c u l t u r e to amino acid starvation.
However,
prolonged exposure to n u t r i t i o n a l l y i n a d e q u a t e c o n d i t i o n s results in
several a d d i t i o n a l alterations in cellular metabolism.
These may be
interpreted as adaptive changes, enabling the cells to s u r v i v e for a
c o n s i d e r a b l e t i m e even though they can no longer proliferate.
A
slowing of the rate of synthesis of ribosomal RNA occurs within 30 to
40 min of amino acid deprivation (Grummt e t a l . ,
1976), consistent
with a reduced cellular requirement for new ribosomes under conditions
where growth does not occur and existing ribosomes are utilized with
reduced efficiency.
Nutritional limitations of many types also result
in a series of co-ordinated metabolic changes termed the 'negative
pJeiotypic growth response' by Hershko e t a.Z. ( 1 9 7 1 ) .
This is
characterized by inhibition of DNA synthesis and reduced transport of
several small precursors into cells, as well as by the translational and
t r a n s c r i p t i o n a l e f f e c t s a l r e a d y discussed.
We also suggest t h a t
e n h a n c e m e n t of p r o t e i n b r e a k d o w n should now be added t o t h i s l i s t ,
since a m i n o acid s t a r v a t i o n s t i m u l a t e s p r o t e o l y s i s in s e v e r a l t y p e s of
a l . , 1976; N e e l y e t a l . ,
1977; F u l k s e t a . Z . ,
cell ( G u n n e t
1975).
In t h e a b s e n c e of an e x o g e n o u s e s s e n t i a l a m i n o a c i d ,
d e g r a d a t i o n of cellular p r o t e i n s can provide a l i m i t e d q u a n t i t y of this
c o m p o u n d s u f f i c i e n t to m a i n t a i n a basal r a t e of p r o t e i n synthesis ( t h e
s t a r v e d cell r a t e shown in Fig. 1).
This m a y be a d e q u a t e f o r t h e
s u r v i v a l of t h e c e l l at least for a short time, until better
n u t r i t i o n a l conditions a r e e n c o u n t e r e d .
Conclusions
In this review we have concentrated on the biochemical behaviour
of tumour cell lines grown in tissue culture.
The implications of the
f i n d i n g s w h i c h we have described may, h o w e v e r , be reasonably
extended to normal tissues in intact animals.
Despite h o m e o s t a t i c
m e c h a n i s m s w h i c h m a i n t a i n n u t r i e n t concentrations in the blood at
nearly constant values, an organ such as the liver has to cope w i t h
w i d e v a r i a t i o n s in the r a t e of i n f l u x of amino acids and o t h e r
compounds from the p o r t a l vein, a s s o c i a t e d w i t h changes in food
intake.
It may be expected that such metabolic challenges are dealt
with by mechanisms at least related to those briefly described in this
article.
Acknowledgements
We are g r a t e f u l to Sigrid B u r r i d g e , Vivienne Tilleray, and Ann
Bloyce for s k i l l f u l t e c h n i c a l assistance and to Dave P e r r e t t (St.
Bartholomew's
H o s p i t a l M e d i c a l U n i t ) for HPLC analyses of
nucleotides.
This work has been supported by grants from the Cancer Research
Campaign and the Medical Research Council. M.3.C is grateful to the
Cancer Research Campaign for a Career Development Award.
REGULATION
OF
PROTEIN
SYNTHESIS
#3
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