AbstractPuromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues

Hipkiss, A.R.

Wharton, S.A.

Elsevier - Publisher Connector 

Volume 168, number 1 FEBS 1296 March 1984 
Abnormal proteins of shortened length are preferentially 
degraded in the cytosol of cultured MRCS fibroblasts 
S.A. Wharton and A.R. Hipkiss 
Department of Blochemlstry, King’s College, Unwersrty of London, Strand, London WC2R 2LS, England 
Received 28 December 1983; revised version received 30 January 1984 
Puromycyl peptrdes were degraded m MRCS ftbroblasts more rapidly than normal proteins labelled for 
the corresponding length of time for both long and short labelling periods. The degradation of the 
puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of 
normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal 
proteins were degraded m the lysosomes. The rapid degradation of the puromycyl peptides was not due 
to the inhibition of protein synthesis brought about by puromycm but was due to the structure of the 
substrates themselves. The degree and intracellular site of degradation of puromycyl peptrdes closely mimrc 
those of abnormal (missense) proteins containing amino acid analogues. 
Puromycin MRC5 flbroblast 
1. INTRODUCTION 
It is important for the cell to be able to remove 
potentially deleterious aberrant molecules. Two 
such abnormal molecules which have been studied 
are proteins with either missense or nonsense er- 
rors, models of which are, respectively, proteins 
containing amino acid analogues and proteins 
prematurely terminated by the action of 
puromycin. It is known that puromycyl proteins 
are rapidly degraded in Escherichia coli [1,2], 
reticulocytes [3-51, and other mammalian cells 
[6,7]. However little work has been carried out on 
the site and mechanism of degradation. Our 
laboratory has established that the ability to 
degrade puromycyl peptides decreases with 
reticulocyte maturation 181. It was shown in [6] 
that degradation varies with the growth state in 
3T3 fibroblasts, not because of differences in the 
degradative machinery but because different pep- 
tides are synthesised and degraded according to the 
growth phase. Short half-life proteins are preferen- 
tially degraded in the cytosol of mammalian cells 
[9,10]. Abnormal proteins containing amino acid 
Proteolysis Lysosome 
analogues are also rapidly degraded and are 
preferentially broken down in the cytosol of 
cultured cells [9,10]. These findings have been ex- 
tended by authors in [ 1 l] who have established that 
even when abnormal proteins of longer half-lives 
than some normal proteins are generated, they are 
still preferentially degraded in the cytosol. This 
means that the site of degradation is not wholly 
dependent upon the half-life of the protein but 
upon the configurational nature of the proteins. 
Here we report that similar conditions apply for 
the degradation of puromycyl peptides of various 
half-lives. 
2. MATERIALS AND METHODS 
2.1. Chemicals 
Methylamine, puromycin dihydrochloride, cyc- 
loheximide and sodium fluoride were purchased 
from Sigma (Poole), phosphate-buffered saline 
tablets (Dulbecco A) were obtained from Oxoid 
(Basingstoke) and L-[4,5-3H]leucine (spec. act. 
64 Ci/mmol) from Amersham International, 
Amersham. All tissue culture materials were pur- 
134 
Publrshed by Elsevrer Scrence Publishers B. V 
00145793/84/$3 00 0 1984 Federation of European Blochemxal Socletles 
Volume 168, number 1 FEBS LETTERS March 1984 
chased from Flow Laboratories, Irvine, as were 
Linbro ‘Space-Saver’ multiwell plates in which the 
degradation experiments were carried out. 
2.2. Methods 
Cell culturing was performed as in [l l] using 
confluent cells of passage numbers of between 30 
and 40. Measurement of protein degradation was 
also as in [ll] with the following differences: 
[‘Hlleucine was used as a radiolabel instead of 
valine with appropriate changes in the labelling 
(leucine-free) and degradation (containing 10 mM 
non-radioactive leucine) media. Separate ex- 
periments have shown that both radiolabels gave 
very similar results (unpublished). For the prepara- 
tion of endogenous puromycyl peptides, cells were 
labelled with t3H]leucine for either 30 min or 18 h 
in the presence of either 10 or 2.5 ,ug/ml 
puromycin dihydrochloride, respectively. Amino 
acid incorporation was measured, correcting for 
cell number, as in [12]. 
3. RESULTS 
3.1. Degradation of long- and short-labelled 
proteins and puromycyl peptides 
Fig.1 shows the percentage degradation of 
30 min and 18 h labelled normal proteins and 
puromycyl peptides in normal medium containing 
a 10% (v/v) supplement of foetal calf serum, and 
in serum-free medium. By prolonging the labelling 
period proteins with longer half-lives tend to be 
preferentially labelled [13] and this is shown in 
fig. 1 with the longer-labelled normal proteins hav- 
ing longer half-lives in serum-containing medium 
(a) than short-labelled normal proteins (c). Pro- 
longing the labelling period also increases the half- 
life of puromycyl peptides so that the half-life of 
the long-labelled puromycyl peptides (b) is similar 
to that of the short-labelled normal proteins (c). 
The increase in the degree of proteolysis brought 
about by the incubation with puromycin was 
similar for long- and short-labelled peptides (2.63- 
and 2.62-fold, after 5 h of degradation). The con- 
centrations of puromycin used, 2.5 ,ug/ml for 
long-labelled proteins, and lOpg/ml for short- 
labelled proteins, were chosen because they in- 
hibited the incorporation of [3H]leucine into pro- 
tein by the same amount, approx. 70%. Neither 
60 (a) 
40 
20 
k/' 
15 24 
25 
1' 5 24 
25 
1. 5 24 __ 25 
Incubation Time (hl " 
Fig.1. The degradation of long-labelled and short- 
labelled normal proteins and puromycyl peptides in 
serum-containing and serum-free media. Approx. 2 x 
10’ cells were labelled for 30 min (short label) or 18 h 
(long label) with [‘Hlleucine in 0.5 ml leucine-free 
Eagles minimal essential medium supplemented with 
10% (v/v) foetal calf serum and 20 mM Hepes buffer 
with or without puromycin (2.5 /rg/ml long label; 
lOpg/ml short label). Degradation was measured over 
24 h in high (10 mM) leucme MEM with and without 
serum and expressed as a percentage total radioactivity 
which is trichloroacetrc acid soluble. (0) Serum- 
contaming medium, (A) serum-free medium. Results are 
means of 3 determinations f SD. (a) Long-labelled 
normal proteins, (b) long-labelled puromycyl peptides, 
(c) short-labelled normal proteins, (d) short-labelled 
puromycyl peptides. 
concentration had any effect upon cell viability as 
determined by trypan blue exclusion (not shown). 
3.2. The degree of lysosomal proteolysis as 
determined by an increase in degradation in 
serum-free medium 
When fibroblasts are placed in serum-free 
135 
Volume 168, number 1 FEBS LETTERS March 1984 
medium the lysosomal contribution to degradation 
is increased [14], thus the extent of the increase in 
proteolysis will give an indication of the degree of 
lysosomal involvement in the degradation. Normal 
long-labelled proteins (fig.la) have the largest 
lysosomal involvement in their degradation. Nor- 
mal short-labelled proteins and long-labelled 
puromycyl peptides show (fig. lc,b) very similar 
(small) amounts of lysosomal involvement as 
determined by this method. Short-labelled 
puromycyl peptides show negligible lysosomal in- 
volvement in their degradation (fig.ld). The 
possibility that proteolysis of the puromycyl pep- 
tides is partly lysosomal in serum-containing 
medium is not excluded by this experiment (but see 
section 3.3). 
3.3. The degree of Iysosomal proteolysis as 
determined by the inhlbition by methylamine 
Another method of assessing lysosomal 
degradation is by use of lysosomotropic agents 
which accumulate in lysosomes, raising their pH 
and thus inactivating those lysosomal proteinases 
which have acid pH optima [ 151. Therefore the 
greater the inhibition brought about by these 
agents, the greater is the lysosomal contribution to 
degradation. The lysosomotropic agent used in this 
study was 10 mM methylamine. Table 1 shows the 
percentage inhibition brought about by 
methylamine compared to controls in serum- 
containing medium and serum-free medium for the 
various classes of labelled proteins. For normal 
long-labelled proteins the inhibition was greater in 
serum-free medium than in serum-containing 
medium which was to be expected as the increase 
in protein degradation brought about by serum- 
free medium is lysosomal in origin. After 24 h in- 
cubation methylamine suppressed egradation to 
similar levels in serum-free medium and serum- 
containing medium for normal long-labelled pro- 
teins (i.e., to 22.8 and 24.7070, respectively) and 
normal short-labelled proteins (to 40.0 and 38.69’0, 
respectively). The percentage inhibition is less for 
short-labelled normal proteins than long-labelled 
normal proteins, showing an increased lysosomal 
contribution in the degradation of the latter. The 
results for both species of puromycyl peptides, on 
the other hand, show very little inhibition by 
methylamine and in fact a slight stimulation is 
observed in some cases. Therefore, as judged by 
this criterion, puromycyl peptides show much less 
lysosomal involvement in their degradation than 
normal proteins in both the serum-stimulated and 
serum-deprived states. 
3.4. The effect of protein syntheses mhlbitors 
during the labellmg period 
Puromycin is a protein synthesis inhibitor, 
therefore the amount of labelled polypeptide 
generated during the labelling period would be 
decreased. To check that the increase in the 
degradation of puromycyl peptides was not due to 
a decreased substrate : enzyme ratio, but due to ac- 
tual alterations in the substrate molecules 
Table 1 
Inhibition of degradation of normal proteins and puromycyl peptides by 10 mM methylamme 
% Inhibition of degradation 
Normal protein Puromycyl peptides 
5h 24h Sh 24 h 
Long-labelled proteins 
Serum-containing medium 
Serum-free medium 
18.5 21.3 3.1 5.8 
32.4 36.9 -9.6 7.1 
Short-labelled proteins 
Serum-containing medium 
Serum-free medium 
17.0 17.7 - 11.5 - 1.1 
21.3 28.8 -4.1 9.4 
Experimental details are as described in the text. Results are means of two determinations. A 
negative value denotes a stimulation of protem degradation 
136 
Volume 168, number 1 FEBS LETTERS March 1984 
60 (al ibl 
Fig.2. The degradation of long-labelled proteins which 
were labelled in the presence of puromycin and 
cycloheximide. Protocol was the same as m fig.1 for 
long-labelled proteins. (a) Control, (*) 2.5 pg/ml 
puromycin, (A) 1.2 FM cycloheximide. Results are 
means of 3 determinations f SD. (a) Serum-containing 
medium, (b) serum-free medium. 
themselves, the effects of cycloheximide, an 
elongation in~bitor, upon protein degradation 
were tested. Puromycin inhibited the incorpora- 
tion of [3H]leucine by 73% in the long-labelled ex- 
periments. In separate experiments it was found 
that 1.2 FM cycloheximide inhibited incorporation 
by 72% and thus this concentration was used in 
subsequent experiments. Proteins were labelled for 
18 h in the presence of either puromycin or 
cycloheximide, and the extent of protein degrada- 
tion was measured as in previous experiments. 
Fig.2 shows that puromycin increased protein 
degradation to a much greater extent than 
cyclohe~mide, particul~ly at shorter incubation 
(chase) times. This effect was greater in serum-free 
medium than in serum-containing medium. 
Puromycin and cycloheximide had no effect upon 
cell viability as determined by trypan blue 
exclusion. 
4. DISCUSSION 
These results show that, in agreement with 
[l-7], puromycyl peptides are degraded faster 
than normal proteins labelled for the correspon- 
ding length of time. To our knowledge, this is the 
first time such an observation has been made with 
NRC5 cells. By altering the labelling period it was 
possible to label preferentia~y puromycyl peptides 
and normal proteins of different half-lives. The 
percentage increase in degradation brought about 
by the action of puromycin was similar in long- 
and short-labelled proteins. The same has been 
found to be the case with abnormal proteins 
brought about by the inclusion of canavanine in- 
stead of arginine [1 1 J. The results with serum-free 
medium and with methylamine show that 
puromycyl peptides are degraded to a greater ex- 
tent in the cytosol than normal proteins labelled 
for the same length of time. The results with 
methylamine and, to a lesser extent serum-free 
medium, indicate that, even when the half-lives of 
the proteins are similar as is the case with normal 
short-labelled and long-labelled puromycyl pep- 
tides, a greater proportion of the puromycyl pep- 
tides are degraded in the cytosol. The increase in 
the percentage degradation brought about by 
puromycin was not due to the protein synthesis in- 
hibitory effects of puromycin which could have 
resulted in a decrease in the synthesis of potential 
substrate and an increase in the degradation rate of 
any substrate produced simply as a consequence of 
a decrease in the substrate : enzyme ratio. It seems 
therefore that the rapid degradation of the 
puromycyl peptides is due to their abnormal struc- 
ture. This is borne out by the finding that in 
reticulocyte lysates, where substrate and enzyme 
con~ntrations can be controlled, puromy~l pep- 
tides of globin are degraded rapidly [4,5]. The dif- 
ferent cellular locations of the degradation of 
puromycyl peptides and normal proteins, as well as 
canavanine-containing proteins and normal pro- 
teins [ 111, indicate there are differences in the pro- 
teolytic pathways of abnormal and normal pro- 
teins. However, in our studies we are only measur- 
ing the rate-limiting step of proteolysis and many 
steps could be common to both pathways. Dif- 
ferences in the steps of the degradation of amino 
acid brogue-cont~ning proteins, and the activity 
of certain proteinases towards short peptides have 
been detected in the reticulocyte [4,&S] and E. co/i 
PI. 
ACKNOWLEDGEMENTS 
S.A.W. thanks the Science and Engineering 
Research Council for financial support. We wish 
to thank Dr P.A. Riley for the gift of stock 
137 
Volume 168, number 1 FEBS LETTERS March 1984 
cultures of the MRC 5 cells, and the University of 
London Central Research Fund for financial help 
with consumables. 
WI 
[71 
@I 
191 
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138 


Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts 

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Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts

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