Download Reduced Temperature Can Block Different Glycoproteins at Different

J. gen. Virol. (1986), 67, 2029-2035.
Printed in Great Britain
2029
Key words: Sendai virus/glycoproteins/transport pathways~temperature effect
Reduced Temperature Can Block Different Glycoproteins at Different
Steps during Transport to the Plasma Membrane
By G E N E V I I ~ V E M O T T E T , C H R I S T I N E T U F F E R E A U AND
LAURENT ROUX*
Microbiology Department, University of Geneva Medical School, C.M.U., 9 avenue de Champel,
1211 Geneva 4, Switzerland
(Accepted 6 June 1986)
SUMMARY
Reduced temperature has been shown to block the cell surface expression of Sendai
virus haemagglutinin-neuraminidase (HN) and fusion (F0) glycoproteins at different
steps of their intracellular transport. At 20 °C, HN was confined to the rough
endoplasmic reticulum or cis Golgi compartment, while Fo acquired complete
resistance to digestion by endo-fl-N-acetylglucosaminidase-H and therefore was
blocked at a more distal location in the pathway of cell surface expression. The
significance of these results for different pathways of transport to the cell surface is
discussed.
Membrane glycoproteins are synthesized by rough endoplasmic reticulum (RER) membranebound ribosomes and are subsequently transported via the Golgi apparatus to the cell surface
(Sabatini et al., 1982). During this transport, the glycoproteins undergo various modifications.
The high mannose glycans, added cotranslationally in the RER, are trimmed and converted to
complex sugars (Hubbard & Ivatt, 1981). In addition, proteins can be modified by acylation or
proteolysis (Schmidt, 1982; Homma & Ohuchi, 1973; Scheid & Choppin, 1974).
To establish precisely the sites at which these various modifications take place, one would
ideally like to block the processing of the glycoproteins at definite biochemical steps and at
defined intracellular sites, to be able to correlate directly the biochemical protein composition
and intracellular location. To achieve this goal different approaches are currently used.
Drugs preventing high mannose sugar addition, like tunicamycin (Takatsuki et al., 1975), or
blocking the transport of the glycoprotein from the Golgi onwards, like monensin (Tartakoff,
1983), are widely used. Tunicamycin which blocks high mannose sugar addition is useful in
defining the role of glycosylation in glycoprotein transport, and drugs like monensin allow the
correlation between an intracellular location and a particular step in glycoprotein processing.
Drugs, however, have the major drawback of their possible direct effect on the cellular
machinery involved in transport.
Another approach is to generate mutant cell lines deficient in enzymes involved in the
glycosylation processing, and to follow the transport and properties of the glycoproteins in such
cell lines (Vischer & Hughes, 1981).
The availability of a series of exo- and endoglycosidases with defined substrate requirements
also facilitates the analysis of glycoproteins isolated at different stages of their transport and are
useful in defining, for instance, the rate of transport through the Golgi apparatus (Kobata, 1979;
Koide & Marumatsu, 1974; Tarentino & Maley, 1974; Elder & Alexander, 1982).
As well as the methods cited above, a very interesting and simple one has been reported by
Matlin & Simons (1983). They observed that influenza virus haemagglutinin was not detected at
the surface of infected cells within 2 h of its synthesis when the cells were incubated at 20 °C.
This contrasted with the surface appearance of the glycoprotein within 15 min during
incubation at 37 °C. At 20 °C, however, terminal glycosylation of the protein was taking place,
0000-7213 O 1986 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 09 Aug 2017 21:12:01
2030
Short communication
suggesting that the block in cell surface expression occurred distal to the Golgi apparatus,
presumably between the Golgi complex and the plasma membrane. Supporting this
interpretation was the quick externalization of the haemagglutinin when the incubation
temperature was raised from 20 to 37 °C. Incubation at 20 °C thus appears to represent a way of
blocking glycoprotein at a specific step of its transport.
To analyse whether the results obtained with the influenza virus haemagglutinin would apply
more generally, we decided to investigate the effect of 20 °C incubation on the cell surface
expression of the Sendai virus haemagglutinin-neuraminidase (HN) and fusion (Fo)
glycoproteins. HN and F0 are viral surface glycoproteins with mol. wt. of 67000 (67K) and 65K
respectively. They both contain N-linked glycans which constitute 9 ~ and 15~ of their
respective final protein mass (Choppin & Compans, 1975; Kohama et al., 1978 ; Yoshima et al.,
1981). While F0 is anchored in the membrane by its C-terminus (Blumberg et al., 1985a), HN
has its N-terminus spanning the membrane (Blumberg et al., 1985b; Hsu & Choppin, 1984). The
rate of transport (at 37 °C) of the two proteins to the cell plasma membrane differs: Fo appears at
the surface with a half-life of about 15 rain, and HN with a half-life of 45 min (Blumberg et al.,
1985b). The native mature structure of the two proteins as estimated by their ability to react with
antibodies is generated at different rates also. F0 reacts fully with antibodies raised against its
mature native form soon after its synthesis and addition of high mannose glycans. H N
immunoreactivity, on the other hand, matures with a half-life of about 30 min after completion
of its synthesis and addition of high mannose sugars. This maturation step is presumably taking
place in the RER and/or the cis Golgi (Mottet et al., 1986). In consequence, apart from
extending the results of M atlin & Simons (1983) to other glycoproteins, this study also compares
the effect of 20 °C incubation on the transport of membrane glycoproteins exhibiting different
characteristics.
In order to study the effect of reduced temperature on HN and Fo cell surface expression,
Sendai virus-infected BHK cells were pulse-labelled with [35S]methionine for 15 min at 37 °C
and then chased for increasing periods of time at 37 or 20 °C. At the end of the chase times, the
extent of cell surface expression of HN and F0 was estimated as described in the legend to Fig. 1.
At 37 °C, a significant amount of F0 (about 40 }/o)was already seen at the cell surface at the end of
the pulse (Fig. 1a, b). This amount increased during subsequent incubation at 37 °C to reach a
maximum level by 20 to 30 min of chase. HN, however, was expressed at the cell surface much
more slowly; not detected after the 15 min pulse, HN gradually reached the surface with a halflife of about 40 to 45 min. These results have been presented more extensively before (Blumberg
et al., 1985 b). At 20 °C, on the other hand, the degree of both F0 and H N surface expression seen
during the chase did not significantly exceed that observed at the end of the 15 min pulse. This
demonstrates that incubation at 20 °C efficiently limits the cell surface expression of F0 and HN.
In fact, incubation at 20 °C drastically slows down the rate of H N and Fo transport to the cell
surface rather than blocks it. After 2 h of chase at 20 °C, about 55 to 6 0 ~ ofF0 and about 30~o of
H N was at the surface and by 16 h most of the HN was eventually seen at the surface (data not
shown).
To define at which intracellular location the proteins are restricted in their transport to the
surface, pulse-labelled HN and Fo were chased at 37 or 20 °C as in Fig. 1, isolated by
immunoprecipitation after cell disruption (total cell immunoprecipitation) and analysed for
their sensitivity to endo-fl-N-acetylglucosaminidase-H (endo-H). Endo-H cleaves the high
mannose sugars [(gluc)g-(man)o-(GlucNAc)z] from the protein backbone before they are
processed to [(man4)-(GlucNAc)2], at which point the sugars become resistant to cleavage
(Tarentino & Maley, 1974). As this trimming takes place in the ER up to the cis Golgi
compartment, sensitivity to endo-H reflects RER or cis Golgi localization of the glycoprotein
and resistance reflects its transport from the RER to more distal compartments of the Golgi
(Hubbard & Ivatt, 1981 ; Tarentino & Maley, 1974). As shown in Fig. 2, HN as well as Fo were
totally sensitive to endo-H at the end of the 15 min pulse at 37 °C (0 h, Fig. 2a, b). After 1 h of
chase at 37 °C about 50 ~o of HN and 100 ~o of Fo had acquired resistance, and after 2 h of chase
about 80~ of HN was resistant. These different rates of acquisition of endo-H resistance at
37 °C which correlate with the different rates of cell surface expression have been noticed
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 09 Aug 2017 21:12:01
Short communication
(a)
2031
20 °C
37 °C
IF
I
V
0
20
60
I
20
60
p--
HNq
F 0 --
Np m
Mq
1.0
8o
(b)
I
•
lj
0.8
Iii /
"#, 0.6
~o.4~,
I
0
I
.u .
. . . . .
- /
/
. . . . . . . . . . .
[]
0.2
0
I
I
20
60
Time of chase (min)
Fig. 1. Cell surface expression o f H N (O, II) and F0 (O, I-q)at 20 °C. BHK cell samples grown in 9 cm diam.
Petri dishes were infected with Sendal virus (m.o.i. of 40). Eighteen h post-infection, the infected cells were
pulse-labelled for 15 min at 37 °C with [35S]methionine, chased at 37 °C (O, 0 ) or 20 °C (Vq, II) for the times
(min) indicated with cold methionine (10 mr,l) and then reacted in situ with Sendai virus antiserum. The cells
were then washed with phosphate-buffered saline to remove excess antibody and solubilized in RIPA buffer
and the immune complexes were recovered with the aid of Staphylococcus aureus beads as previously
described (Blumberg et al., 1985a, b). Identical samples of each cell sample were then separated by SDSPAGE. (a) Autoradiograph of the gel; lane V, viral protein markers. (b) Scanning of the autoradiograph to
show the amount of protein recovered at the cell surface expressed as a function of chase time and
temperature.
previously (Mottet et al., 1986). At 20 °C, even if the rate of acquisition of endo-H resistance for
Fo was decreased (only about 70 % resistant after 1 h of chase), F0 nevertheless reached complete
resistance within 2 h of chase. This contrasts with HN which remained totally sensitive to endoH at 20 °C during the same period of observation. These results suggest that, at 20 °C, HN
remains completely in the RER or the cis Golgi compartment while Fo is efficiently transported
from the RER to the Golgi. As the apparent molecular weight of Fo analysed after 2 h of chase at
20 °C corresponds to that of the mature protein, Fo presumably undergoes terminal sugar
processing.
To assess the RER localization of HN during the 20 °C incubation, the degree of maturation
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 09 Aug 2017 21:12:01
2032
Short communication
37 °C
20 °C
1
'1 I
0
(
V
I
--
1
q-
]
I
--
t
2
+
I I
--
1
+
It
--
2
+
II
--
+
"[
P
HN
F 0
NP
F 1
M
]
H?
F
N]
Fig. 2. Endo-H sensitivity of pulse-labelled and chased HN and Fo. Sendai virus-infected BHK cell
samples were pulse-labelled with [3sS]methionineand chased (0, 1 or 2 h) as in the experiment shown in
Fig. 1. The HN and F 0 proteins, recovered by total cell immunoprecipitation using monoclonal
antibodies (Roux et al., 1984), were resuspended in 1~ SDS, 50 mM-Tris-HC1 pH 6.8, 0.5~ 2mercaptoethanol, 2 mM-phenylmethylsulphonylfluoride and boiled for 5 min. After a 10-fold dilution
with 125 mM-sodium citrate pH 5-0, aliquots of the proteins were digested overnight at 37 °C with
80 mU/ml endo-H (Nenzymes) (+) or mock-treated (-). The proteins were then concentrated by
acetone precipitation (8 : 1, v/v) and analysed by PAGE. (a) HN ; (b) F0. Lane V, viral protein markers.
of H N native immunoreactivity was controlled. H N immunoreactivity has been shown to
mature during the first hour following the synthesis of the protein (Mottet et al., 1986). This
maturation step takes place in the R E R or at the most in the cis Golgi compartment. It was thus
of interest to analyse whether H N immunoreactivity would mature at 20°C. The
immunoreactivity of H N for a monoclonal antibody raised against the native mature form of the
molecule was therefore estimated as described in detail elsewhere (Mottet et al., 1986) and the
results of these estimations are shown in Fig. 3. In this experiment, the infected cells are pulselabelled and chased as in Fig. 1 and the H N is recovered by total immunoprecipitation as in Fig.
2. At 37 °C, H N immunoreactivity matured.during the first hour following its synthesis as
evidenced by the increased efficiency at which the protein was recovered by immunoprecipitation. This maturation did not take place at 20 °C. This experiment thus confirms that H N is
blocked in the R E R at low temperature. As expected, F0 which shows no immunoreactivity
maturation (Mottet et al., 1986) is not affected in its immunoreactivity by incubation at low
temperature.
Therefore, if incubation at 20 °C efficiently prevents cell surface expression of Sendai virus
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 09 Aug 2017 21:12:01
Short communication
(a)
HN
F0
i
I I
37 °C
20 °C
I
V
2033
I I
0
20
60
37 °C
20 °C
II
20
60
[I
0
20
60
20
60
p--
HN-F 0 --
NP--
M--
(b)
1.0
I
I
~- . . . . .
/ "O~
~
~
•
~
. . . . . . . . . . .
-~--1~
8 0.8
0-6
.
o= 0.4
0.2
0
I
I
20
60
Time of chase (min)
Fig. 3. Immunoreactivity of pulse-labelled and chased HN (O, I ) and Fo (©, El). Sendai virusinfected BHK cell samples were pulse-labelled and chased for the times (min) indicated at 37 °C (O, O)
or 20 °C (I-q, I ) as described in the legend to Fig. 1. HN and Fo were then recovered by total cell
immunoprecipitation from identical aliquots of cellular extracts using anti-HN (S-16) or anti-F0 (M16) monoclonal antibodies as described in detail in Mottet et al. (1986). The immunoprecipitates were
then analysed by PAGE. (a) Autoradiograph of the gel; lane V, viral protein markers. (b) Scanning of
the autoradiograph to show the amount of protein recovered expressed as a function of chase time and
temperature.
H N and F0 glycoproteins, it appears to affect the intracellular transport of the two proteins in
different ways. H N transport is efficiently blocked in the R E R or the cis Golgi compartment. Fo
transport, on the other hand, can still proceed until the protein becomes totally resistant to endoH, but is arrested before cell surface expression. In conclusion, this study confirms and extends
the observation of Matlin & Simons (1983). Incubation at 20 °C appears to be a reliable method
to block cell surface expression of m e m b r a n e glycoproteins. The step at which the proteins are
blocked, however, may differ from protein to protein.
The differential effect of reduced temperature in restricting H N and Fo is yet another factor
which can be a d d e d to the list o f differences already reported, namely in the oligosaccharide
patterns, in the rate of cell surface expression, in the orientation of anchorage in the p l a s m a
m e m b r a n e and finally a difference in the maturation process of their native mature
immunoreactivity (Yoshima et al., 1981 ; Blumberg et al., 1985 a, b; Mottet et al., 1986). This list
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 09 Aug 2017 21:12:01
2034
Short commun&ation
of different properties exhibited by HN and Fo questions the processing pathway(s) that the two
glycoproteins follow to mature and to reach the cell surface. Classically, HN and F0 would follow
one unique pathway, and the differences observed between the two proteins would only reflect
intrinsic properties linked to each protein. Thus, the different rate in the cell surface expression
could result from the difference in the orientation of anchorage in the membrane which basically
depends on the primary structure of the protein. The different effect of reduced temperature in
restricting HN and F o cellular transport could in this case only result from the difference in the
rate of transport. If transport from RER or cis Golgi to the more distal compartments of the
Golgi is more sensitive to low temperature than transport from the middle Golgi onwards, then
the transport of the fast moving F0 protein could be less affected since F0 could possibly reach
the middle Golgi before the effect of low temperature is present. This interpretation does not
satisfactorily account for the different patterns of glycosylation as already mentioned by
Yoshima et al. (1981) and for the evolution of F0 endo-H resistance at 20 °C observed here.
Alternatively, HN and F0 could follow different pathways with different glycosylation
machinery, different rates of transport and different sensitivities to reduced temperature.
Although the different characteristics of HN and F0 do not constitute a formal proof of the
existence of such different pathways, they certainly indicate that this possibility has to be
seriously considered.
We are grateful to Dr Allen Portner for providing us with the monoclonal antibodies. This work was supported
by a grant no. 3. 332.0.82 from the Fonds National Suisse de la Recherche Scientifique.
REFERENCES
BLUMBERG, B. M., GIORGI, C., ROSE, K. & KOLAKOFSKY,D. (1985a). Sequence determination of Sendal virus fusion
protein gene. Journal of General Virology 66, 317-331.
BLUMBERG,B. M., GIORGI, C., ROUX, L., RAJU, R., DOWLING, P., CHOLLET, A. & KOLAKOFSKY,D. (1985b). Sequence
determination of the Sendal virus HN gene and its comparison to the influenza virus glycoproteins. Cell 41,
269-276.
CHOPPIN, P. W. & COMPANS,R. W. (1975). Reproduction of Paramyxoviruses. In Comprehensive Virology, vol. 4, pp.
95-178. Edited by H. Fraenkel-Conrat & R. R. Wagner. New York & London: Plenum Press.
ELDER, L H. & ALEXANDER,S. (1982). Endo-fl-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium
meningosepticum that cleaves both high-mannose and complex glycoproteins. Proceedings of the National
Academy of Sciences, U.S.A. 79, 4540-4544.
HOMMA,M. & OHUCHI, M. (1973). Trypsin action on the growth of Sendal virus in tissue culture cells. III. Structural
difference of Sendal viruses grown in eggs and in tissue culture cells. Journal of Virology 12, 1457-1465.
HSU, M. C. & CHOt'PIN, P. W. (1984). Analysis of Sendal virus m R N A s with c D N A clones of viral genes and
sequences of biologically important regions of the fusion protein. Proceedingsof the National Academy of
Sciences, U.S.A. 81, 7732-7736.
HUBBARD,S. C. & IVATT,R. J. (1981). Synthesis and processing of asparagine-linked oligosaccharides. Annual Review
of Biochemistry 50, 555-583.
KOBATA, A. (1979). Use of endo- and exoglycosidases for structural studies of glycoconjugates. Analytical
Biochemistry 100, 1-14.
KOHAMA,T., SHIMIZU,K. & ISHIDA, N. 0978). Carbohydrate composition of the envelope glycoproteins of Sendal
virus. Virology 90, 226-234.
KOIDE, N. & MARUMATSU, T. (1974). Endo-fl-N-acetylglucosaminidase acting on carbohydrate moieties of
glycoproteins. Journal of Biological Chemistry 249, 4897-4904.
MATLIN, K. S. & SIMONS,K. (1983). Reduced temperature prevents transfer of a membrane glycoprotein to the cell
surface but does not prevent terminal glycosylation. Cell 34, 233-243.
MOTTET, G., PORTNER, A. & ROUX, L. (1986). Drastic immunoreactivity changes between the immature and mature
forms of the Sendal virus HN and F 0 glycoproteins. Journal of Virology 59, 132-141.
ROUX, L., BEFFY, P. & PORTNER, A. (1984). Restriction of cell surface expression of Sendal virus hemagglutininneuraminidase glycoprotein correlates with its higher instability in persistently and standard plus defective
interfering virus infected BHK-21 cells. Virology 138, 118-128.
SABATINI, D. D., KREIBICH, G., MORIMOTO,T. & MILTON, A. 0982). Mechanisms for the incorporation of proteins in
membranes and organelles. Journal of Cell Biology 92, 1-22.
SCHEID, A. & CHOt't'IN, P. W. (1974). Identification of the biological activities of paramyxovirus glycoproteins.
Activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of
Sendal virus. Virology 57, 457-490.
SCltMIDT, M. F. G. (1982). Acylation of viral spike glycoproteins: a feature of enveloped R N A viruses. Virologyl l 6 ,
327-338.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 09 Aug 2017 21:12:01
Short communication
2035
TAKATSUKI,A., KOHRO,K. & TAMURA,G. (1975). Inhibition of biosynthesis of polyisoprenol sugars in chick embryo
microsomes by tunicamycin. Agricultural and Biological Chemistry 39, 2089-2091.
TARENTINO, A. L. & MALEY,F. (1974). Purification and properties of an endo-beta-N-acetylglucosaminidasefrom
Streptomyces gorisens. Journal of Biological Chemistry 249, 811-817.
TARTAKOFF,A. (1983). Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell 32, 10261028.
VISCHER,P. & HUGHES,R. C. (1981). Glycosyl transferases of baby-hamster-kidney (BHK) cells and ricin-resistant
mutants. European Journal of Biochemistry 117, 275-284.
YOSHIMA, H., NAKANISHI,M., OKADA, Y. & KOBATA,A. (1981). Carbohydrate structures of HVJ (Sendai virus)
glycoproteins. Journal of Biological Chemistry 256, 5355-5361.
(Received 18 April 1986)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 09 Aug 2017 21:12:01