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Selective and transient association of sendai virus HN glycoprotein with BiP
VIROLOGY
175,161-166(1990)
Selective and Transient
Association
of Sendai Virus HN Glycoprotein
with BiP
LAURENT ROUX Department
of Microbiology,
University of Geneva Medical School, C. M. U., 9Avenue Received July 17, 1989; accepted November
de Champel,
12 11 Geneva 4. Switzerland
6, 1989
From 1 0-min [35S]methionine pulse-labeled Sendai virus-infected BHK cells, an anti-BiP monoclonal antibody precipitated, along with the BiP protein, the hemagglutinin-neuraminidase protein (SV-HN) fivefold better than the fusion protein (SV-Fo). A minimal estimate of 309’0 of the newly made HN was complexed to BiP. The majority of the HN in the complex was endo-H sensitive and the molar ratio of BiP:HN was estimated to be 1:2. With time, HN dissociated from BiP, and the rate of dissociation was found to be inversely proportional to the rate at which HN acquired its native structure. It is proposed that association with BiP followed by slow release (i) is responsible for the HN slow maturation and (ii) represents a normal step in its maturation pathway. 0 1990AcaUemic Press. Inc.
INTRODUCTION
is also reflected in the time course of cell surface expression: Fo and HN reach the cell surface with a halftime of 10 and 30-40 min, respectively (Blumberg et a/., 1985a). No explanation for this delay in SV-HN maturation has been provided so far. lmmunoglobulin (lg) heavy chain binding protein (BiP) is a member of the heat shock protein family. Found identical with the glucose regulated 78-kDa protein (GRP78), it is abundant in the rough endoplasmic reticulum (RER)of normal cells (Pelham, 1986). It was found to irreversibly associate with aberrantly glycosylated or nonglycosylated proteins, with proteins containing incorrect disulfide bonds or with improperly assembled multimeric proteins (Bole et a/., 1986; Dorner et a/,, 1987; Gething eta/., 1986; Haas and Wabl, 1983; Hendershot et a/., 1987; Kassenbrock et al., 1988; Kozutsumi et al., 1988). Because of these associations, and because BiP belongs to the heat shock protein family, it has been proposed that BiP may bind to misfolded proteins to prevent their denaturation or aggregation (Munro and Pelham, 1986; Pelham, 1986; Kassenbrock and Kelly, 1989). On the other hand, if BiP was found to associate tightly and irreversibly with lg heavy chains in nonproducing myeloma cell lines, in antibody producing cell lines, the BiP/lg heavy chain complex was found transient and was disrupted by heavy and light chain association (Haas and Wabl, 1983; Bole et al., 1986). It has therefore also been proposed that BiP participate in protein folding (Pelham, 1986; Gething et al., 1986). Recent published data support, indeed, this role for BiP and for another member of the heat shock protein family, the hsp60 protein (Ostermann et al,, 1989; Normington et a/., 1989). In this paperwe provide data that support the participation of BiP in the normal maturation process of the SV-HN protein.
Sendai virus (SV), a member of the paramyxoviridae family, is an enveloped single-stranded RNA genome virus. Its genome encodes six to nine proteins among which are two surface glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion (Fo) proteins (for a recent review, see Kolakofsky and Roux, 1987). SV-HN, which is responsible for the attachment of the virus particle to the target cell, is 576 amino acids long and contains five potential N-linked glycosylation sites (Blumberg era/., 1985a). It only contains one highly hydrophobic domain of 25 amino acids positioned at residues 35-60 from the N-terminus of the protein. Therefore, SV-HN, as the HN of all the members of the paramyxoviridae family, appears to be anchored in the membrane by its N-terminus (Blumberg et al., 1985a). SV-Fo, which, in its activated form, is responsible for the fusion of the viral envelope with the cell membrane, is 565 amino acids long and contains three potential Nlinked glycosylation sites. A signal peptide of 25 amino acids is cleaved from its N-terminus, and a hydrophobic domain of 24 amino acids (residues 500-523), positioned at 42 amino acids from the C-terminus, anchors the protein in the membrane (Blumberg et a/., 1985b). In infected cells, HN and Fo are synthesized in the rough endoplasmic reticulum (RER)and cotranslationally glycosylated at roughly equal rates (Mottet et a/., 1986). They nevertheless acquire their native structure and travel to the plasma membrane at different rates. Fo intramolecular disulfide bond formation and native folding take place rapidly following synthesis (within O5 min). In contrast, for HN, the intramolecular disulfide bonds are formed with a lag of at least 10 min and native folding takes place with a half time of about 30 min (Mottet et a/., 1986; Vidal et al., 1989). This difference 161
0042-6822/90$3.00 Copyright Q 1990 by Academic Press. Inc. All rtghts of reproducton I” any form resewed.
162
LAURENT
MATERIALS
AND METHODS
ROUX
@
Cells and virus
Baby hamster kidney (BHK-21) cells were routinely grown in regular minimum Eagle’s medium (MEM) supplemented with 5% inactivated fetal calf serum (FCS) under 5% CO, atmosphere. Sendai virus (Harris strain) defective interfering particle free stock was prepared in g-day-old embryonated chicken eggs as described before (Roux and Holland, 1979). BHK cells were infected (m.o.i. of about 40) at 33” for 1 hr in MEM and then further incubated at the same temperature in MEM-2% FCS for about 24 hr. Labeling, immunoprecipitation, endoglycosidase H digestion
and
RESULTS Transient
and selective
association
of HN with BiP
To evaluate a possible association of the Sendai virus proteins with BiP, newly synthesized proteins from
-
-
d-&P
-
+-+-+-+ PULSE
v
@
For pulse [35S]methionine labeling, infected cells were preincubated for 30 min in a methionine-free MEM, then labeled for 10 min (see Results) with 150 &i/ml of [35S]methionine. At the end of the pulse, the cells were either collected or further incubated in MEM-2% FCS containing 10 mM cold methionine (chase). For continuous [35S]methionine-labeling experiments, BHK cells, after passage, were grown in MEM-5% FCS in the presence of 20 &i/ml of [35S]methionine until confluence. The cells were then passaged 1:4 and further grown to confluence in the same radioactive medium. When confluent, the cells were infected and further grown for 24 hr in MEM-2% FCS containing [35S]methionine. lmmunoprecipitations were performed as described in detail before (Mottet et a/., 1986) except that 100 mM of iodoacetamide (IAA) was routinely added to the cell lysing buffer to prevent undesirable formation of disulfide bonds during cellular extract preparation. Briefly, the cells were disrupted in Triton-SDS buffer (150 mM NaCI, 1% deoxycholate, 1% Triton X-l 00, 0.1% SDS, 10 mlMTris hydrochloride, pH 7.8, 100 rnM IAA), sonicated, and centrifuged for 15 min in an Eppendorf centrifuge. The supernatants (cellular extracts) were then incubated with the antibodies and the protein A-Sepharose CL-4B. A threefold excess volume of a 50% suspension of protein A-Sepharose over the volume of rabbit antiserum was generally used to ensure quantitative binding of the IgG to the protein A. After washing, the Sepharose beads were resuspended in polyacrylamide gel electrophoresis buffer (PAGE; Laemmli, 1970). Endo-H digestion of the immunoprecipitates were performed as described before (Mottet et a/., 1986).
INFECTED
INFECTED d- HN d- F,
1
2
+ +
-
-
+ +
CHASE
3 4‘ ‘5
6
7 8’
- _ + + +-+-+-+-
_
+ +
-+-+-+-+ PULSE , Vl2345678
CHASE
h i,s.
M --MU@ FIG. 1. Selective association of HN with BiP. Mock or Sendai virusinfected BHK cells were pulse labeled with [35S]methionine for 10 min at 18 hr postinfection or pulse labeled and chased for 45 min. Cellular extracts corresponding to 10’ cells were then incubated with (A) 20 pi of a monoclonal antibody antiimmunoglobulin heavy chain binding protein (anti-BiP, Bole et a/., 1986) or (B) 10 ~1 of an anti-HN or 5 ~1 of an anti Fo monoclonal antibody (respectively, M-9 and M33, Roux et a/., 1984). The immunoprecipitates were then analyzed by PAGE and the autoradiograms were scanned by densitometry to estimate the amounts of proteins. V, viral protein markers with apparent molecularweight according to Lamb eta/. (1976). P, polymeraseassociated protein; HN, hemagglutinin-neuraminidase protein; Fo, fusion protein; NP, nucleocapsid protein; M, matrix protein. BiP, immunoglobulin heavy chain binding protein.
infected cell extracts were reacted with an anti-BiP monoclonal antibody (Bole et a/., 1986). Figure 1A shows that, with the BiP protein (78 kDa; Bole et al., 1986) coprecipitated two proteins, with a PAGE migration corresponding to that of SV-HN (72 kDa) and SVFo (65 kDa), (Fig. 1A, lane 4). After a 45-min chase period, the amount of the two viral proteins associated with BiP decreased by 1O-fold under conditions where the amount of BiP was basically unchanged (lane 8). Of the two viral proteins associated with BiP, HN was clearly the most prominent. The relative association of HN and Fo with BiP was estimated by comparing their amounts synthesized during the pulse. HN and Fo were recovered by specific monoclonal antibodies (Fig. 1B). These antibodies recognize the native forms of the
SELECTIVE AND TRANSIENT
ASSOCIATION
proteins (Mottet et al., 1986). As the HN native structure is formed after a lag of about 30 min (Mottet et al., 1986) the relative amounts of Fo and HN synthesized during the pulse are indicated by the amounts found in lane 4 (for Fo) and lane 7 (for HN). Such a comparison shows that there was 2.7 times more HN synthesized than Fo. This difference could not account for the 14fold difference, in favor of HN, observed in the association of the two proteins with BiP, as illustrated in Fig. 1A, lane 4. Therefore HN appeared to selectively associate with BiP 5-fold more efficiently than Fo. It is finally noteworthy that infection by SV appeared to increase the synthesis of BiP by a factor of 5, as shown by the comparison of the amounts of BiP in lanes 2 and 4 in Fig. 1.
@
163
OF SV-HN WITH BiP
VI234
5
6
7
P dBiP
Estimation of the amount of newly made HN associated with BiP BiP is a very prevalent protein in the RER (Pelham, 1986) and its association with HN might have been fortuitous. Also, only a minor fraction of HN could have associated with BiP, as appears to be the case for the influenza virus HA protein (Gething et al., 1986). It was therefore important to measure the fraction of the newly made HN complexed to BiP. However, to do so, it was not possible to quantitatively precipitate BiP, because of the limited availability of the anti-BiP antibody. An estimate was rather obtained in the following manner. The amount of newly synthesized HN was estimated by quantitative precipitation using an antibody, Rab-HNsDS,which preferentially recognizes the nonnative (newly made) form of HN (Mottet et a/., 1986; Vidal et al., 1989). This amount was then related to the amount of HN recovered by 20 ~1of anti-BiP antibody. Figure 2A (lanes 1 to 4) shows an example of the immune precipitations, and Fig. 2B shows the quantitation of the HN recovered (curve l ), as well as those of two companion experiments. The plateau reached with 20 and 30 ~1of Rab-HNsDsensured a quantitative recovery of HN. The amount of HN associated with BiP is then shown in Fig. 2A (lane 6) and represented 30%. This value is reported as a hatched rectangle in Fig. 2B. Thirty percent represents a minimum estimate, since no assurance was taken in the experiment to quantitatively precipitate BiP for the reason mentioned above. Therefore, a substantial amount of the newly synthesized HN was found associated with BiP. Estimation of the HN/BiP ratio Anotherway to evaluate the extent of the HN/BiP association was to estimate the molar ratio of the two proteins in the complex. The ratios that can be calculated from data in Figs. 1 and 2 are misleading, since
microliters of anti-HN&b FIG. 2. Minimal estimation of the amount of newly made HN associated with BiP. Sendai virus-infected BHK cells were pulse labeled with [%]methionine for 10 min at 18 hr postinfection or pulse labeled and chased for 45 min. (A) Quantitative HN immune precipitations using 5, 10, 20, and 30 ~1 of Rab-HNsDs antiserum and 100 ~1 of a 50% suspensron of protein A-Sepharose are shown, as well as immune precipitations with anti-P (5 ~1, lane 5) and anti-BiP (20 pl, lanes 6 and 7) monoclonal antibodies using the equivalent of 2 X 1O5cells. For immune precipitation with anti-BiP antibodies pulse-labeled (lane 6) and pulse-chase-labeled (lane 7) cells were used. (B) Three separate quantitative HN immune precipitations using increasing amounts of Rab-HNsDs and cellular extracts from 4 X lo5 (A) or 2 X 1O5 (0, n) pulse-labeled cells, were scanned by densitometry and plotted. Curve 0 represents the experiment shown in A, and curves A and n represent the results of two companion experiments. V. viral protein markers, as in the legend to Fig. 1. In (8) the hatched rectangle refers to the estimation of HN associated with BiP measured from (A) lane 6.
the labeled proteins only reflect the rate of their synthesis during the IO-min pulse, and not their molar amounts. Cells were therefore continuously labeled with [35S]methionine (see Materials and Methods), and immune precipitations were carried out with the RabHNsDsantiserum to detect the amounts of BiP bound to HN. Rab-HNsDs antiserum, as mentioned above, preferentially reacts with the nonnative form of the protein (i.e., the newly made protein), although it binds to mature HN (i.e., virion HN) as well, but with poor efficiency. Figure 3A (lane 2) shows that Rab-HNsDsalso
164
LAURENT
v
1
2 -
BiP
@
0
MO-H:
-
5mu
2odJ
PL
-
5nu
zofru -P
BiP HNf
-HN
GD 1
5mU 2
2QmlJ: Endo-H 3 UC-P
pL BiP?6 -
FIG. 3. Estimation of the HN/BiP ratio and of the HN Endo-H sensitivity. BHK cells continuously labeled with [35S]methionine were mock or Sendai virus infected. After 24 hr of infection, cells were collected and the virus particles produced in the medium purified as described before (Roux and Waldvogel, 1982). Cellular extracts corresponding to 1O7 cells (A, B, D) or virus particles produced by 2 X 1O7 cells (C) were precipitated with 100 pl Rab-HNsDs (A, B, C) or Rab-Fo,,, antiserum (D). Fractions of the immunoprecipitates (l/20) were treated or not with endoglycosydase-H. V, viral protein markers as in legend to Fig. 1.
precipitated a protein whose PAGE migration was identical to that of BiP. That this protein was associated with HN is demonstrated by the fact that BiP is not precipitated from uninfected cells (lane 1). Figure 3B further shows that the majority of HN recovered was sensitive to endo-H digestion. Endo-H sensitivity is characteristic of glycoproteins still residing in the RERresistance to endo-H is indeed acquired after sugar processing taking place in the cis-Golgi (Tarentino and Maley, 1974). Therefore, most of the HN recovered in association with BiP originated from the RER. To demonstrate that HN was capable of acquiring endo-H resistance upon further processing, the minor fraction of virion HN recognized by Rab-HNsDswas analyzed (Fig. 3C). In virions, HN was not associated with BiP (the minor band observed above HN corresponds to P) and was insensitive to endo-H digestion. When immunoprecipitations of the cellular extracts were performed with a Rab-Fosos antiserum (Rab-Fos,,s exhibits for Fo the properties that Rab-HNsDshave for HN; Mottet et al., 1986) only a small amount of BiP was visible, and the majority of the Fo precipitated in this way was resistant to endo-H digestion (Fig. 3D). From the amounts
ROUX
of HN and BiP proteins recovered in two experiments, a molar abundance of 1.58 and 2.06 mol of HN to 1.O mol of BiP was estimated, by taking into account the methionine content of the two proteins (10 and 9 for BiP and HN, respectively; Munro and Pelham, 1986; Blumberg et al., 1985a). These results suggested that about 50% of the HN recovered from the RERcould be associated with BiP, if the molar ratio of the HN/BiP complex was a 1: 1 ratio. If, on the other hand, the complex would be made of 2 HN and 1 BiP, then all the HN in the RER could be bound to BiP. Therefore these experiments suggested that BiP binds to a large fraction of the newly made HN. They also confirmed the limited association of Fo with BiP seen in Figs. 1 and 2. Rate of dissociation of HN from BiP HN acquires its native conformation slowly with a half time of about 30 min (Mottet et a/., 1986). It was therefore of interest to follow the rate of dissociation of HN from BiP. Infected cells were pulse labeled and chased for increasing periods of time. At each time point half the cellular extracts were reacted with a monoclonal which recognizes native HN (Fig. 4A), and half the extracts with the anti-BiP (Fig. 4B). The time courses of HN maturation and of the association with BiP were thus measured in parallel. Figure 4C shows these data graphically after densitometry scanning of the autoradiograms. As reported before (Mottet et al., 1986), HN native immunoreactivity was formed with a half-time of about 30 min. The association of HN with BiP appeared to be maximal after synthesis, and then decreased progressively during 60 min of chase. The half-life of the complex was again about 30 min. The amount of HN associated with BiP appeared inversely proportional to the amount of properly folded or native HN. DISCUSSION Newly made SV-HN is found associated with BiP fivefold more efficiently than is SV-Fo. This selectivity might only reflect the time HN and Fo spend in the RER, since Fo acquires resistance to endo-H twice as fast as HN (Mottet eta/., 1986). Although there is little information concerning this point for normal proteins, the hypothesis that the degree of association with BiP might reflect the time spent in the RER is contradicted by the case of the spleen focus forming virus (SFFV) envelope glycoprotein. SFFV glycoprotein is defective in transport to the cell plasma membrane and is retained in the RER, presumably because of a defect in oligomerization. Despite this retention in the RER, SFFV glycoprotein is not found in association with BiP (Kilpatrick et a/., 1989). Alternatively, the limited
SELECTIVE AND TRANSIENT
@
CHASE TIME v HN-
@
CHASE TIME
Ek-
0' 10' 15' 20' 25' 30' 45' 60' 90' I I I I I I I I I “*c *UI’
.,
v
0' IO' 15' 2U 25' 3p' 45' 60' 90'
I
I
I
I
I
I
I
I
I
HN-
0
0 10 20 30 45 60
90
minutes FIG. 4. Relationship between HN immunoreactivity maturation and HN/BiP association. Sendai virus-infected BHK cell samples (10” cells) were pulse labeled with r5S]methionine and then chased for increasing periods of time. Half the cellular extracts were then incubated with (A) 5 ~1 of anti-HN (M-9) or(B) 3 ~1 of anti-BiP monoclonal antibodies. The immunoprecipitates were then analyzed by PAGE and the autoradiograms were scanned by densitometry to estimate the relative amounts of HN immunoprecipitated and the HN/BiP ratios as a function of the period of chase. (C) Plot of the amounts of immunoprecipitated HN and of the HN/BiP ratios as a function of minutes of chase measured by densitometry scanning of the autoradiograms shown in (A) and(B). V, viral markers.
amount of Fo complexed with BiP may result from short half-life of the Fo/BiP complex. The newly made Fo folds in its mature conformation (evidenced by reactivity with anti-native antibodies) soon after its synthesis, whereas HN requires a half-time of about 30 min (Mottet eta/., 1986; see also Fig. 1B). An attempt to capture a higher percentage of Fo associated with BiP by shortening the [35S]methionine pulse (3 min) partly succeeded, since the ratio of HN/Fo in the BiP complex increased by a factor of 3.5 over that found after a 1Omin pulse (as in Fig. 1). The experiment, however, was disserved by the poor reactivity of the anti-BiP antibody for the BiP/HN and the BiP/Fo complexes, leading to recovery of minute amounts of BiP, Fo, as well as HN proteins under these conditions (not shown). This could indicate that Fo as well efficiently associates with
ASSOCIATION
OF SV-HN WITH BiP
165
BiP after its synthesis. However, more work has to be done to clarify this point. We estimated that a minimum of 30% of the newly made HN was found complexed to BiP (Fig. 2). After continuous labeling of the cells with [35S]methionine, however, a minimum of 50% of Endo-H sensitive HN was found complexed with BiP in a one to one molar ratio (Fig. 3). This high percentage, along with the transitory nature of the HN/BiP association, argues against, but does not absolutely rule out, a participation of this complex in HN degradation. Association with BiP leading to degradation has indeed been found to be irreversible (Bole et al., 1986). Moreover, selective degradation of protein in the RER has been recently shown not to involve association with BiP (Chen et al., 1988). Therefore, the HN/BiP association described here rather supports a participation of BiP in the normal HN maturation process. A transient association of unfolded SV5 HN with BiP has been recently described and claimed as well to reflect a normal step in the glycoprotein maturation (Ng era/., 1989). So far no satisfactory explanation has accounted for the delay in HN maturation (Mottet et a/., 1986; Vidal eta/., 1989). In view of the correlation between the time course of detachment of HN from BiP and that of the formation of the native conformation (Fig. 4), we propose that it is the attachment of HN to BiP followed by a slow release that is responsible for this delay. This timing is also in agreement with the lag observed in the HN intramoleculardisulfide bond formation required for HN native folding (Vidal et a/., 1989). If association with BiP constitutes a necessary step in the maturation of a certain class of glycoproteins, the question of its availability can be raised. The generally accepted abundance of BiP in the RER (Pelham, 1986) should allow its putative function to be fulfilled under normal conditions. Upon viral infection, when the glycoprotein synthesis is potentially increased, induction of BiP synthesis (as suggested by Fig. 1 which shows a fivefold increase in BiP synthesis; see also Peluso et al., 1978) would adequately increase the supply of BiP. ACKNOWLEDGMENTS The author is Indebted to John Kearney who provided the antibody to BiP, to Richard Compans and Daniel Kolakofsky for pertinent discussions and criticisms, and to Genevigve Mottet for excellent technical assistance. This work was supported by grants from WHO and from the Swiss National Foundation for Scientific Research.
REFERENCES BLUMBERG, B. M., GIORGI, C., Roux, L., RAJU, R., DOWLING. P., CHOLLET, A., and KOLAKOFSKY.D. (1985a). Sequence determination of the Sendai virus HN gene and its comparison to the influenza glycoproteins. Cell 41, 269-278.
166
LAURENT ROUX
BLUMBERG, B. M., GIORGI,C., ROSE,K., and KOLAKOFSKY, D. (198513). Sequence determination of the Sendai virus fusion protein gene. J. Gen. Viral. 66, 317-331. BOLE,D. G., HENDERSHOT, L. M., and KEARNY,J. F. (1986). Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. /. Ce//Bio/. 102, 1558-l 566. CHEN,C., BONIFACIO, 1. S., YUAN.L. C., and KLAUSNER, R. D. (1988). Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment. J. Cell Biol. 107. 2 149-2 161. DORNER, A. J., BOLE,D. G., and KAUFMAN,R. J. (1987). The relationship of N-linked glycosylation and heavy chain-binding protein associated with the secretion of glycoproteins. J. Ce// Biol. 105, 26652674. GETHING,M.-J., MCCAMMON,K., and SAMBROOK. 1.(1986). Expression of wild-type and mutant forms of influenza hemagglutinin: The role of folding in intracellular transport. Cell46, 939-950. HAAS,I. G., and WABL,M. (1983). lmmunoglobulin heavy chain binding protein. Nature (London) 306,387-389. HENDERSHOT, L. M., BOLE, D. G., KOHLER,G., and KEARNEY,J. F. (1987). Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain binding protein. J. Cell Biol. 104, 761-767. KASSENBROCK, C. K., GARCIA,P. D., WALTER.P., and KELLY,R. B. (1988). Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature (London) 333, 90-93. KASSENBROCK. C. K., and KELLY,R. B. (1989). Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides. EMBOJ. 8,1461-1467.
KILPATRICK, D. R., SRINIVAS,R. V., and COMPANS,R. W. (1989). The spleen focus-forming virus envelope glycoprotein is defective in oligomerization. J. Biol. Chem. 264, 10,732-l 0.737. KOLAKOFSKY, D., and Roux, L. (1987). The molecular biology of paramyxoviruses. In “The Molecular Basis of Viral Replication” (P. R. Bercoff, Ed.), pp. 277-297. Plenum, New York. KOZUTSUMI, Y., SEGAL,M., NORMINGTON, K., GETHING,M.-J., and SAMBROOK,1. (1988). The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature (London) 332,462-464. LAEMMLI,U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
LAMB,R. A., MAHY,B. W. J., and CHOPPIN,P. W. (1976). The synthesis of Sendai virus polypeptides in infected cells. Virology 69, 116131. MOTTET,G., PORTNER, A., and Roux, L. (1986). Drastic immunoreactivity changes between the immature and mature forms of the Sendai virus HN and Fo glycoproteins. J. Wo/, 59. 132-141. MUNRO,S.. and PELHAM,H. R. B. (1986). An Hsp70-like protein in the RER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell46, 291-300. NG. T. W.. RANDALL,R. E., and LAMB,R. A. (1989). Intracellular maturation and transport of the SV5 type II glycoprotein HN: Specific and transient association with GRP78-BiP i the ER and extensive internalization from the cell surface. J. Ce// Biol,, in press. NORMINGTON, K., KOHNO,K., KOZUTSUMI, Y., GETHING,M.-J., and SAMBROOK, J. (1989). S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell57, 12231236. OSTERMANN, J., HORWICH, A. L., NEUPERT, W., and HARTL,F.-U. (1989). Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Narure (London) 341, 125-l 30. PELHAM,H. R. B. (1986). Speculations on the function of the major heat shock and glucose-regulated proteins. Ce//46,959-961. PELUSO,R. W., LAMB,R. A., and CHOPPIN,P. W. (1978). Infection with paramyxoviruses stimulates synthesis of cellular polypeptides that are also stimulated in cells transformed by Rous sarcoma virus or deprived in glucose. Proc. Nat/. Acad. Sci. USA 75,6120-6124. Roux, L., and HOLLAND,1.J. (1979). Role of defective interfering particles of Sendai virus in persistent infections. Virology 93,91-l 03. Roux, L., and WALVOGEL, F. A. (1982). Instability of viral M protein in BHK-21 cells persistently infected with Sendai virus. Cell 28, 293302. Roux, L., BEFFY,P., and PORTNER,A. (1984). Restriction of Sendai virus hemagglutinin-neuraminidase glycoprotein correlates with its higher instability in persistently and standard plus defective interfering virus infected BHK-21 cells. Virology 138, 118-l 28. TARENTINO, A. L., and MALEY,F. (1974). Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. 1. Biol. Chem. 249,81 l-81 7. VIDAL,S., MOTTET,G., KOLAKOFSKY, D., and Roux, L. (1989). Addition of high-mannose sugars must precede disulfide bond formation for proper folding of Sendai virus glycoproteins. J. Viral. 63, 892900.
175,161-166(1990)
Selective and Transient
Association
of Sendai Virus HN Glycoprotein
with BiP
LAURENT ROUX Department
of Microbiology,
University of Geneva Medical School, C. M. U., 9Avenue Received July 17, 1989; accepted November
de Champel,
12 11 Geneva 4. Switzerland
6, 1989
From 1 0-min [35S]methionine pulse-labeled Sendai virus-infected BHK cells, an anti-BiP monoclonal antibody precipitated, along with the BiP protein, the hemagglutinin-neuraminidase protein (SV-HN) fivefold better than the fusion protein (SV-Fo). A minimal estimate of 309’0 of the newly made HN was complexed to BiP. The majority of the HN in the complex was endo-H sensitive and the molar ratio of BiP:HN was estimated to be 1:2. With time, HN dissociated from BiP, and the rate of dissociation was found to be inversely proportional to the rate at which HN acquired its native structure. It is proposed that association with BiP followed by slow release (i) is responsible for the HN slow maturation and (ii) represents a normal step in its maturation pathway. 0 1990AcaUemic Press. Inc.
INTRODUCTION
is also reflected in the time course of cell surface expression: Fo and HN reach the cell surface with a halftime of 10 and 30-40 min, respectively (Blumberg et a/., 1985a). No explanation for this delay in SV-HN maturation has been provided so far. lmmunoglobulin (lg) heavy chain binding protein (BiP) is a member of the heat shock protein family. Found identical with the glucose regulated 78-kDa protein (GRP78), it is abundant in the rough endoplasmic reticulum (RER)of normal cells (Pelham, 1986). It was found to irreversibly associate with aberrantly glycosylated or nonglycosylated proteins, with proteins containing incorrect disulfide bonds or with improperly assembled multimeric proteins (Bole et a/., 1986; Dorner et a/,, 1987; Gething eta/., 1986; Haas and Wabl, 1983; Hendershot et a/., 1987; Kassenbrock et al., 1988; Kozutsumi et al., 1988). Because of these associations, and because BiP belongs to the heat shock protein family, it has been proposed that BiP may bind to misfolded proteins to prevent their denaturation or aggregation (Munro and Pelham, 1986; Pelham, 1986; Kassenbrock and Kelly, 1989). On the other hand, if BiP was found to associate tightly and irreversibly with lg heavy chains in nonproducing myeloma cell lines, in antibody producing cell lines, the BiP/lg heavy chain complex was found transient and was disrupted by heavy and light chain association (Haas and Wabl, 1983; Bole et al., 1986). It has therefore also been proposed that BiP participate in protein folding (Pelham, 1986; Gething et al., 1986). Recent published data support, indeed, this role for BiP and for another member of the heat shock protein family, the hsp60 protein (Ostermann et al,, 1989; Normington et a/., 1989). In this paperwe provide data that support the participation of BiP in the normal maturation process of the SV-HN protein.
Sendai virus (SV), a member of the paramyxoviridae family, is an enveloped single-stranded RNA genome virus. Its genome encodes six to nine proteins among which are two surface glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion (Fo) proteins (for a recent review, see Kolakofsky and Roux, 1987). SV-HN, which is responsible for the attachment of the virus particle to the target cell, is 576 amino acids long and contains five potential N-linked glycosylation sites (Blumberg era/., 1985a). It only contains one highly hydrophobic domain of 25 amino acids positioned at residues 35-60 from the N-terminus of the protein. Therefore, SV-HN, as the HN of all the members of the paramyxoviridae family, appears to be anchored in the membrane by its N-terminus (Blumberg et al., 1985a). SV-Fo, which, in its activated form, is responsible for the fusion of the viral envelope with the cell membrane, is 565 amino acids long and contains three potential Nlinked glycosylation sites. A signal peptide of 25 amino acids is cleaved from its N-terminus, and a hydrophobic domain of 24 amino acids (residues 500-523), positioned at 42 amino acids from the C-terminus, anchors the protein in the membrane (Blumberg et a/., 1985b). In infected cells, HN and Fo are synthesized in the rough endoplasmic reticulum (RER)and cotranslationally glycosylated at roughly equal rates (Mottet et a/., 1986). They nevertheless acquire their native structure and travel to the plasma membrane at different rates. Fo intramolecular disulfide bond formation and native folding take place rapidly following synthesis (within O5 min). In contrast, for HN, the intramolecular disulfide bonds are formed with a lag of at least 10 min and native folding takes place with a half time of about 30 min (Mottet et a/., 1986; Vidal et al., 1989). This difference 161
0042-6822/90$3.00 Copyright Q 1990 by Academic Press. Inc. All rtghts of reproducton I” any form resewed.
162
LAURENT
MATERIALS
AND METHODS
ROUX
@
Cells and virus
Baby hamster kidney (BHK-21) cells were routinely grown in regular minimum Eagle’s medium (MEM) supplemented with 5% inactivated fetal calf serum (FCS) under 5% CO, atmosphere. Sendai virus (Harris strain) defective interfering particle free stock was prepared in g-day-old embryonated chicken eggs as described before (Roux and Holland, 1979). BHK cells were infected (m.o.i. of about 40) at 33” for 1 hr in MEM and then further incubated at the same temperature in MEM-2% FCS for about 24 hr. Labeling, immunoprecipitation, endoglycosidase H digestion
and
RESULTS Transient
and selective
association
of HN with BiP
To evaluate a possible association of the Sendai virus proteins with BiP, newly synthesized proteins from
-
-
d-&P
-
+-+-+-+ PULSE
v
@
For pulse [35S]methionine labeling, infected cells were preincubated for 30 min in a methionine-free MEM, then labeled for 10 min (see Results) with 150 &i/ml of [35S]methionine. At the end of the pulse, the cells were either collected or further incubated in MEM-2% FCS containing 10 mM cold methionine (chase). For continuous [35S]methionine-labeling experiments, BHK cells, after passage, were grown in MEM-5% FCS in the presence of 20 &i/ml of [35S]methionine until confluence. The cells were then passaged 1:4 and further grown to confluence in the same radioactive medium. When confluent, the cells were infected and further grown for 24 hr in MEM-2% FCS containing [35S]methionine. lmmunoprecipitations were performed as described in detail before (Mottet et a/., 1986) except that 100 mM of iodoacetamide (IAA) was routinely added to the cell lysing buffer to prevent undesirable formation of disulfide bonds during cellular extract preparation. Briefly, the cells were disrupted in Triton-SDS buffer (150 mM NaCI, 1% deoxycholate, 1% Triton X-l 00, 0.1% SDS, 10 mlMTris hydrochloride, pH 7.8, 100 rnM IAA), sonicated, and centrifuged for 15 min in an Eppendorf centrifuge. The supernatants (cellular extracts) were then incubated with the antibodies and the protein A-Sepharose CL-4B. A threefold excess volume of a 50% suspension of protein A-Sepharose over the volume of rabbit antiserum was generally used to ensure quantitative binding of the IgG to the protein A. After washing, the Sepharose beads were resuspended in polyacrylamide gel electrophoresis buffer (PAGE; Laemmli, 1970). Endo-H digestion of the immunoprecipitates were performed as described before (Mottet et a/., 1986).
INFECTED
INFECTED d- HN d- F,
1
2
+ +
-
-
+ +
CHASE
3 4‘ ‘5
6
7 8’
- _ + + +-+-+-+-
_
+ +
-+-+-+-+ PULSE , Vl2345678
CHASE
h i,s.
M --MU@ FIG. 1. Selective association of HN with BiP. Mock or Sendai virusinfected BHK cells were pulse labeled with [35S]methionine for 10 min at 18 hr postinfection or pulse labeled and chased for 45 min. Cellular extracts corresponding to 10’ cells were then incubated with (A) 20 pi of a monoclonal antibody antiimmunoglobulin heavy chain binding protein (anti-BiP, Bole et a/., 1986) or (B) 10 ~1 of an anti-HN or 5 ~1 of an anti Fo monoclonal antibody (respectively, M-9 and M33, Roux et a/., 1984). The immunoprecipitates were then analyzed by PAGE and the autoradiograms were scanned by densitometry to estimate the amounts of proteins. V, viral protein markers with apparent molecularweight according to Lamb eta/. (1976). P, polymeraseassociated protein; HN, hemagglutinin-neuraminidase protein; Fo, fusion protein; NP, nucleocapsid protein; M, matrix protein. BiP, immunoglobulin heavy chain binding protein.
infected cell extracts were reacted with an anti-BiP monoclonal antibody (Bole et a/., 1986). Figure 1A shows that, with the BiP protein (78 kDa; Bole et al., 1986) coprecipitated two proteins, with a PAGE migration corresponding to that of SV-HN (72 kDa) and SVFo (65 kDa), (Fig. 1A, lane 4). After a 45-min chase period, the amount of the two viral proteins associated with BiP decreased by 1O-fold under conditions where the amount of BiP was basically unchanged (lane 8). Of the two viral proteins associated with BiP, HN was clearly the most prominent. The relative association of HN and Fo with BiP was estimated by comparing their amounts synthesized during the pulse. HN and Fo were recovered by specific monoclonal antibodies (Fig. 1B). These antibodies recognize the native forms of the
SELECTIVE AND TRANSIENT
ASSOCIATION
proteins (Mottet et al., 1986). As the HN native structure is formed after a lag of about 30 min (Mottet et al., 1986) the relative amounts of Fo and HN synthesized during the pulse are indicated by the amounts found in lane 4 (for Fo) and lane 7 (for HN). Such a comparison shows that there was 2.7 times more HN synthesized than Fo. This difference could not account for the 14fold difference, in favor of HN, observed in the association of the two proteins with BiP, as illustrated in Fig. 1A, lane 4. Therefore HN appeared to selectively associate with BiP 5-fold more efficiently than Fo. It is finally noteworthy that infection by SV appeared to increase the synthesis of BiP by a factor of 5, as shown by the comparison of the amounts of BiP in lanes 2 and 4 in Fig. 1.
@
163
OF SV-HN WITH BiP
VI234
5
6
7
P dBiP
Estimation of the amount of newly made HN associated with BiP BiP is a very prevalent protein in the RER (Pelham, 1986) and its association with HN might have been fortuitous. Also, only a minor fraction of HN could have associated with BiP, as appears to be the case for the influenza virus HA protein (Gething et al., 1986). It was therefore important to measure the fraction of the newly made HN complexed to BiP. However, to do so, it was not possible to quantitatively precipitate BiP, because of the limited availability of the anti-BiP antibody. An estimate was rather obtained in the following manner. The amount of newly synthesized HN was estimated by quantitative precipitation using an antibody, Rab-HNsDS,which preferentially recognizes the nonnative (newly made) form of HN (Mottet et a/., 1986; Vidal et al., 1989). This amount was then related to the amount of HN recovered by 20 ~1of anti-BiP antibody. Figure 2A (lanes 1 to 4) shows an example of the immune precipitations, and Fig. 2B shows the quantitation of the HN recovered (curve l ), as well as those of two companion experiments. The plateau reached with 20 and 30 ~1of Rab-HNsDsensured a quantitative recovery of HN. The amount of HN associated with BiP is then shown in Fig. 2A (lane 6) and represented 30%. This value is reported as a hatched rectangle in Fig. 2B. Thirty percent represents a minimum estimate, since no assurance was taken in the experiment to quantitatively precipitate BiP for the reason mentioned above. Therefore, a substantial amount of the newly synthesized HN was found associated with BiP. Estimation of the HN/BiP ratio Anotherway to evaluate the extent of the HN/BiP association was to estimate the molar ratio of the two proteins in the complex. The ratios that can be calculated from data in Figs. 1 and 2 are misleading, since
microliters of anti-HN&b FIG. 2. Minimal estimation of the amount of newly made HN associated with BiP. Sendai virus-infected BHK cells were pulse labeled with [%]methionine for 10 min at 18 hr postinfection or pulse labeled and chased for 45 min. (A) Quantitative HN immune precipitations using 5, 10, 20, and 30 ~1 of Rab-HNsDs antiserum and 100 ~1 of a 50% suspensron of protein A-Sepharose are shown, as well as immune precipitations with anti-P (5 ~1, lane 5) and anti-BiP (20 pl, lanes 6 and 7) monoclonal antibodies using the equivalent of 2 X 1O5cells. For immune precipitation with anti-BiP antibodies pulse-labeled (lane 6) and pulse-chase-labeled (lane 7) cells were used. (B) Three separate quantitative HN immune precipitations using increasing amounts of Rab-HNsDs and cellular extracts from 4 X lo5 (A) or 2 X 1O5 (0, n) pulse-labeled cells, were scanned by densitometry and plotted. Curve 0 represents the experiment shown in A, and curves A and n represent the results of two companion experiments. V. viral protein markers, as in the legend to Fig. 1. In (8) the hatched rectangle refers to the estimation of HN associated with BiP measured from (A) lane 6.
the labeled proteins only reflect the rate of their synthesis during the IO-min pulse, and not their molar amounts. Cells were therefore continuously labeled with [35S]methionine (see Materials and Methods), and immune precipitations were carried out with the RabHNsDsantiserum to detect the amounts of BiP bound to HN. Rab-HNsDs antiserum, as mentioned above, preferentially reacts with the nonnative form of the protein (i.e., the newly made protein), although it binds to mature HN (i.e., virion HN) as well, but with poor efficiency. Figure 3A (lane 2) shows that Rab-HNsDsalso
164
LAURENT
v
1
2 -
BiP
@
0
MO-H:
-
5mu
2odJ
PL
-
5nu
zofru -P
BiP HNf
-HN
GD 1
5mU 2
2QmlJ: Endo-H 3 UC-P
pL BiP?6 -
FIG. 3. Estimation of the HN/BiP ratio and of the HN Endo-H sensitivity. BHK cells continuously labeled with [35S]methionine were mock or Sendai virus infected. After 24 hr of infection, cells were collected and the virus particles produced in the medium purified as described before (Roux and Waldvogel, 1982). Cellular extracts corresponding to 1O7 cells (A, B, D) or virus particles produced by 2 X 1O7 cells (C) were precipitated with 100 pl Rab-HNsDs (A, B, C) or Rab-Fo,,, antiserum (D). Fractions of the immunoprecipitates (l/20) were treated or not with endoglycosydase-H. V, viral protein markers as in legend to Fig. 1.
precipitated a protein whose PAGE migration was identical to that of BiP. That this protein was associated with HN is demonstrated by the fact that BiP is not precipitated from uninfected cells (lane 1). Figure 3B further shows that the majority of HN recovered was sensitive to endo-H digestion. Endo-H sensitivity is characteristic of glycoproteins still residing in the RERresistance to endo-H is indeed acquired after sugar processing taking place in the cis-Golgi (Tarentino and Maley, 1974). Therefore, most of the HN recovered in association with BiP originated from the RER. To demonstrate that HN was capable of acquiring endo-H resistance upon further processing, the minor fraction of virion HN recognized by Rab-HNsDswas analyzed (Fig. 3C). In virions, HN was not associated with BiP (the minor band observed above HN corresponds to P) and was insensitive to endo-H digestion. When immunoprecipitations of the cellular extracts were performed with a Rab-Fosos antiserum (Rab-Fos,,s exhibits for Fo the properties that Rab-HNsDshave for HN; Mottet et al., 1986) only a small amount of BiP was visible, and the majority of the Fo precipitated in this way was resistant to endo-H digestion (Fig. 3D). From the amounts
ROUX
of HN and BiP proteins recovered in two experiments, a molar abundance of 1.58 and 2.06 mol of HN to 1.O mol of BiP was estimated, by taking into account the methionine content of the two proteins (10 and 9 for BiP and HN, respectively; Munro and Pelham, 1986; Blumberg et al., 1985a). These results suggested that about 50% of the HN recovered from the RERcould be associated with BiP, if the molar ratio of the HN/BiP complex was a 1: 1 ratio. If, on the other hand, the complex would be made of 2 HN and 1 BiP, then all the HN in the RER could be bound to BiP. Therefore these experiments suggested that BiP binds to a large fraction of the newly made HN. They also confirmed the limited association of Fo with BiP seen in Figs. 1 and 2. Rate of dissociation of HN from BiP HN acquires its native conformation slowly with a half time of about 30 min (Mottet et a/., 1986). It was therefore of interest to follow the rate of dissociation of HN from BiP. Infected cells were pulse labeled and chased for increasing periods of time. At each time point half the cellular extracts were reacted with a monoclonal which recognizes native HN (Fig. 4A), and half the extracts with the anti-BiP (Fig. 4B). The time courses of HN maturation and of the association with BiP were thus measured in parallel. Figure 4C shows these data graphically after densitometry scanning of the autoradiograms. As reported before (Mottet et al., 1986), HN native immunoreactivity was formed with a half-time of about 30 min. The association of HN with BiP appeared to be maximal after synthesis, and then decreased progressively during 60 min of chase. The half-life of the complex was again about 30 min. The amount of HN associated with BiP appeared inversely proportional to the amount of properly folded or native HN. DISCUSSION Newly made SV-HN is found associated with BiP fivefold more efficiently than is SV-Fo. This selectivity might only reflect the time HN and Fo spend in the RER, since Fo acquires resistance to endo-H twice as fast as HN (Mottet eta/., 1986). Although there is little information concerning this point for normal proteins, the hypothesis that the degree of association with BiP might reflect the time spent in the RER is contradicted by the case of the spleen focus forming virus (SFFV) envelope glycoprotein. SFFV glycoprotein is defective in transport to the cell plasma membrane and is retained in the RER, presumably because of a defect in oligomerization. Despite this retention in the RER, SFFV glycoprotein is not found in association with BiP (Kilpatrick et a/., 1989). Alternatively, the limited
SELECTIVE AND TRANSIENT
@
CHASE TIME v HN-
@
CHASE TIME
Ek-
0' 10' 15' 20' 25' 30' 45' 60' 90' I I I I I I I I I “*c *UI’
.,
v
0' IO' 15' 2U 25' 3p' 45' 60' 90'
I
I
I
I
I
I
I
I
I
HN-
0
0 10 20 30 45 60
90
minutes FIG. 4. Relationship between HN immunoreactivity maturation and HN/BiP association. Sendai virus-infected BHK cell samples (10” cells) were pulse labeled with r5S]methionine and then chased for increasing periods of time. Half the cellular extracts were then incubated with (A) 5 ~1 of anti-HN (M-9) or(B) 3 ~1 of anti-BiP monoclonal antibodies. The immunoprecipitates were then analyzed by PAGE and the autoradiograms were scanned by densitometry to estimate the relative amounts of HN immunoprecipitated and the HN/BiP ratios as a function of the period of chase. (C) Plot of the amounts of immunoprecipitated HN and of the HN/BiP ratios as a function of minutes of chase measured by densitometry scanning of the autoradiograms shown in (A) and(B). V, viral markers.
amount of Fo complexed with BiP may result from short half-life of the Fo/BiP complex. The newly made Fo folds in its mature conformation (evidenced by reactivity with anti-native antibodies) soon after its synthesis, whereas HN requires a half-time of about 30 min (Mottet eta/., 1986; see also Fig. 1B). An attempt to capture a higher percentage of Fo associated with BiP by shortening the [35S]methionine pulse (3 min) partly succeeded, since the ratio of HN/Fo in the BiP complex increased by a factor of 3.5 over that found after a 1Omin pulse (as in Fig. 1). The experiment, however, was disserved by the poor reactivity of the anti-BiP antibody for the BiP/HN and the BiP/Fo complexes, leading to recovery of minute amounts of BiP, Fo, as well as HN proteins under these conditions (not shown). This could indicate that Fo as well efficiently associates with
ASSOCIATION
OF SV-HN WITH BiP
165
BiP after its synthesis. However, more work has to be done to clarify this point. We estimated that a minimum of 30% of the newly made HN was found complexed to BiP (Fig. 2). After continuous labeling of the cells with [35S]methionine, however, a minimum of 50% of Endo-H sensitive HN was found complexed with BiP in a one to one molar ratio (Fig. 3). This high percentage, along with the transitory nature of the HN/BiP association, argues against, but does not absolutely rule out, a participation of this complex in HN degradation. Association with BiP leading to degradation has indeed been found to be irreversible (Bole et al., 1986). Moreover, selective degradation of protein in the RER has been recently shown not to involve association with BiP (Chen et al., 1988). Therefore, the HN/BiP association described here rather supports a participation of BiP in the normal HN maturation process. A transient association of unfolded SV5 HN with BiP has been recently described and claimed as well to reflect a normal step in the glycoprotein maturation (Ng era/., 1989). So far no satisfactory explanation has accounted for the delay in HN maturation (Mottet et a/., 1986; Vidal eta/., 1989). In view of the correlation between the time course of detachment of HN from BiP and that of the formation of the native conformation (Fig. 4), we propose that it is the attachment of HN to BiP followed by a slow release that is responsible for this delay. This timing is also in agreement with the lag observed in the HN intramoleculardisulfide bond formation required for HN native folding (Vidal et a/., 1989). If association with BiP constitutes a necessary step in the maturation of a certain class of glycoproteins, the question of its availability can be raised. The generally accepted abundance of BiP in the RER (Pelham, 1986) should allow its putative function to be fulfilled under normal conditions. Upon viral infection, when the glycoprotein synthesis is potentially increased, induction of BiP synthesis (as suggested by Fig. 1 which shows a fivefold increase in BiP synthesis; see also Peluso et al., 1978) would adequately increase the supply of BiP. ACKNOWLEDGMENTS The author is Indebted to John Kearney who provided the antibody to BiP, to Richard Compans and Daniel Kolakofsky for pertinent discussions and criticisms, and to Genevigve Mottet for excellent technical assistance. This work was supported by grants from WHO and from the Swiss National Foundation for Scientific Research.
REFERENCES BLUMBERG, B. M., GIORGI, C., Roux, L., RAJU, R., DOWLING. P., CHOLLET, A., and KOLAKOFSKY.D. (1985a). Sequence determination of the Sendai virus HN gene and its comparison to the influenza glycoproteins. Cell 41, 269-278.
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BLUMBERG, B. M., GIORGI,C., ROSE,K., and KOLAKOFSKY, D. (198513). Sequence determination of the Sendai virus fusion protein gene. J. Gen. Viral. 66, 317-331. BOLE,D. G., HENDERSHOT, L. M., and KEARNY,J. F. (1986). Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. /. Ce//Bio/. 102, 1558-l 566. CHEN,C., BONIFACIO, 1. S., YUAN.L. C., and KLAUSNER, R. D. (1988). Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment. J. Cell Biol. 107. 2 149-2 161. DORNER, A. J., BOLE,D. G., and KAUFMAN,R. J. (1987). The relationship of N-linked glycosylation and heavy chain-binding protein associated with the secretion of glycoproteins. J. Ce// Biol. 105, 26652674. GETHING,M.-J., MCCAMMON,K., and SAMBROOK. 1.(1986). Expression of wild-type and mutant forms of influenza hemagglutinin: The role of folding in intracellular transport. Cell46, 939-950. HAAS,I. G., and WABL,M. (1983). lmmunoglobulin heavy chain binding protein. Nature (London) 306,387-389. HENDERSHOT, L. M., BOLE, D. G., KOHLER,G., and KEARNEY,J. F. (1987). Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain binding protein. J. Cell Biol. 104, 761-767. KASSENBROCK, C. K., GARCIA,P. D., WALTER.P., and KELLY,R. B. (1988). Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature (London) 333, 90-93. KASSENBROCK. C. K., and KELLY,R. B. (1989). Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides. EMBOJ. 8,1461-1467.
KILPATRICK, D. R., SRINIVAS,R. V., and COMPANS,R. W. (1989). The spleen focus-forming virus envelope glycoprotein is defective in oligomerization. J. Biol. Chem. 264, 10,732-l 0.737. KOLAKOFSKY, D., and Roux, L. (1987). The molecular biology of paramyxoviruses. In “The Molecular Basis of Viral Replication” (P. R. Bercoff, Ed.), pp. 277-297. Plenum, New York. KOZUTSUMI, Y., SEGAL,M., NORMINGTON, K., GETHING,M.-J., and SAMBROOK,1. (1988). The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature (London) 332,462-464. LAEMMLI,U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
LAMB,R. A., MAHY,B. W. J., and CHOPPIN,P. W. (1976). The synthesis of Sendai virus polypeptides in infected cells. Virology 69, 116131. MOTTET,G., PORTNER, A., and Roux, L. (1986). Drastic immunoreactivity changes between the immature and mature forms of the Sendai virus HN and Fo glycoproteins. J. Wo/, 59. 132-141. MUNRO,S.. and PELHAM,H. R. B. (1986). An Hsp70-like protein in the RER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell46, 291-300. NG. T. W.. RANDALL,R. E., and LAMB,R. A. (1989). Intracellular maturation and transport of the SV5 type II glycoprotein HN: Specific and transient association with GRP78-BiP i the ER and extensive internalization from the cell surface. J. Ce// Biol,, in press. NORMINGTON, K., KOHNO,K., KOZUTSUMI, Y., GETHING,M.-J., and SAMBROOK, J. (1989). S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell57, 12231236. OSTERMANN, J., HORWICH, A. L., NEUPERT, W., and HARTL,F.-U. (1989). Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Narure (London) 341, 125-l 30. PELHAM,H. R. B. (1986). Speculations on the function of the major heat shock and glucose-regulated proteins. Ce//46,959-961. PELUSO,R. W., LAMB,R. A., and CHOPPIN,P. W. (1978). Infection with paramyxoviruses stimulates synthesis of cellular polypeptides that are also stimulated in cells transformed by Rous sarcoma virus or deprived in glucose. Proc. Nat/. Acad. Sci. USA 75,6120-6124. Roux, L., and HOLLAND,1.J. (1979). Role of defective interfering particles of Sendai virus in persistent infections. Virology 93,91-l 03. Roux, L., and WALVOGEL, F. A. (1982). Instability of viral M protein in BHK-21 cells persistently infected with Sendai virus. Cell 28, 293302. Roux, L., BEFFY,P., and PORTNER,A. (1984). Restriction of Sendai virus hemagglutinin-neuraminidase glycoprotein correlates with its higher instability in persistently and standard plus defective interfering virus infected BHK-21 cells. Virology 138, 118-l 28. TARENTINO, A. L., and MALEY,F. (1974). Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. 1. Biol. Chem. 249,81 l-81 7. VIDAL,S., MOTTET,G., KOLAKOFSKY, D., and Roux, L. (1989). Addition of high-mannose sugars must precede disulfide bond formation for proper folding of Sendai virus glycoproteins. J. Viral. 63, 892900.