Studies on compartmentation and turnover of murine retrovirus envelope proteins

VIROLOGY 188, 477-485 (1992)

Studies on Compartmentation and Turnover of Murine Retrovirus Envelope Proteins

Y. Yu AND f’. K. Y. WONG’

The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville. Texas 78957

Received November 26, 199 1; accepted February 11, 1992

Several aspects of turnover and degradation of cell membrane proteins were studied in an NIH 3T3 cell clone expressing the env gene of Moloney murine leukemia virus tsl. Both internalization and shedding of the extracellular domain of the envelope protein gp70 occurred at the cell surface, albeit, in the case of shedding, only a very small fraction of gp70 was shed. The turnover rate of gp70 at the cell surface was similar to that of the same protein in the postendoplasmic reticulum intracellular compartment. In the presence of L-methionine methyl ester, the transmembrane domain of the envelope protein Prpl5E was degraded faster than gp70. o 1992 Academic PESS, I~C.

INTRODUCTION

The biosynthesis of retroviral envgene products has been extensively studied as a component of the retrovirus or as a model system of cell membrane proteins (Earl et a/., 1991; Fitting and Kabat, 1982; Hare, 1989; Kamps et a/., 1991; Hunter and Swanstrom, 1990; Wil- ley et a/., 1988). The general outline of envelope protein biosynthesis of retroviruses is already known. For example, in murine leukemia virus, the envelope glycoproteins, like those of other retroviruses, are synthesized in the endoplasmic reticulum (ER) as a single polypeptide precursor, gPr80e”“, which is rapidly glycosylated. The newly synthesized gPr80e”” begins to fold soon after biosynthesis and forms oligomers in the ER (Kamps et a/., 1991). The process of oligomerization appears to occur very rapidly after biosynthesis, and a series of chase experiments conducted in our labora- tory indicated that trimerization of the gPr80e”” is evi- dent by a 15-min chase (Kamps et al., 1991). Although oligomerization of monomeric subunits is important for transport from the ER to the Golgi apparatus, oligomerization alone does not automatically qualify oligomeric molecules for transport. For efficient transport, correct folding and configuration of the oligomer is required. In the Golgi apparatus, gPr80”“” is post-translationally modified with O-linked oligosaccharides (Pinter and Honnen, 1988) which occurs prior to its cleavage by a host cell protease (Fitting and Kabat, 1982) to form two subunits: the larger gp70 extracellular domain, SU, and the smaller Prpl5E transmembrane domain, TM. In the briefly radiolabeled cells, gp70 and Prpl5E can be detected after a 30-min chase (Kamps et a/., 1991). The glycosylated gp70/Prpl5E complex is then trans-

’ Author to whom correspondence and reprint requests should be addressed.

ported out of the Golgi network to the cell surface. All of these processes occur whether or not other viral gene products are present and whether or not virions are being assembled (Schultz and Rein, 1985). Upon assembly of the virion, Prpl5E is further processed to pl5E and p2E by the viral-coded protease (Henderson et al., 1984).

Despite the progress that has been made in under- standing the biosynthesis and processing of retroviral envelope proteins, relatively little is known about the behaviors of the envelope proteins after they reach the cell surface. In the study reported here, several aspects of the envelope protein turnover in post-ER intracellular compartments, as well as at the cell surface, were examined using a cell line that expresses only the envelope proteins of tsl as a tool. tsl is a temperature-sensitive mutant of Moloney murine leukemia virus (MoMuLV) (Wong et al., 1973). The struc- ture and functions of tsl env gene and its products have been well characterized (Wong, 1990; Wong and Yuen, 1991; Yuen et al., 1985, 1986). A single amino acid change in gPr80e”” at position 25 has been shown to be responsible for the temperature sensitivity of tsl (Szurek et al., 1990). At the restrictive temperature (39.5”) the mutant oligomers of gPr80e”“are defective in transport from the ER to the Golgi (Kamps et a/., 1991). As a result, gPr80e”” is not processed to gp70 and Prpl5E and remains endoglycosidase H sensitive (Szurek et a/., 1990). To facilitate studies on the turnover of the envelope proteins, an expression system containing the tsl env gene alone has been con- structed, and a clonal cell line, clone 1, which expresses the env gene of tsl through the expression system, has been established (Vu et al., 199 1). Since there is no gag gene product in clone 1 cells, the cell was shown not to release anyvirions. Furthermore, the large amounts of envelope proteins expressed in clone

477 0042-6822192 $5.00 CopyrIght 0 1992 by Academic Press, Inc. All rights of reproduction I” any form reserved

478 YU AND WONG

1 cells retained the temperature-sensitive phenotype of the tsl virus (Vu eta/., 1991). Because of the temperature-sensitive phenotype of the tsl envelope proteins, a “temperature shift” experiment can be exploited to study the turnover of a limited amount of envelope proteins in the post-ER intracellular compartment and at the cell surface. Radiolabeled gPr80”“” can be trapped in the ER upon biosynthesis at 39.5” and the trapped proteins can be released simultaneously by a temperature shift down to 34”. Transport of gPr80e”’ would proceed from the ER to the Golgi to be processed to gp70andPrpl5E.Afterashortperiodofchaseat34”, the cells will be shifted back to 39.5” and kept at this temperature for the remaining chase period in order to block export of newly synthesized gPr80”““from the ER at the late stage of chase. This procedure is followed so that the fate of the gPr80e”” that has already pro- ceeded from the ER to the Golgi can be determined.

The studies reported here showed that after gp70 reached the cell surface both internalization and shedding of this protein occurred although only a very small amount of shedding of gp70 was observed. We also found that both the gp70 located at the cell surface and in the post-ER intracellular compartment have a similar turnover rate. Furthermore, when L-methionine methyl ester was added to the clone 1 cell tissue culture medium, Prpl5E was observed to be degraded faster than gp70.

MATERIALS AND METHODS

Cells and viruses

ptsl-env(F)-transfected NIH 3T3 clone 1 cell line (clone 1) (Vu et al., 1991) NIH 3T3 cells infected with MoMuLV tsl virus (Wong, 1990) and MoMuLV ts3 virus (Yuen and Wong, 1977) were described previously. psi-2 cells were obtained from Dr. R. Mulligan (Mann et a/., 1983). All cell lines were maintained in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) supplemented with 6% fetal calf serum and 4% newborn calf serum.

lodination of the cell surface gp70

lodinations were done 12 hr after the plating of 1.5 X lo6 clone 1 cells in 60-mm-diameter tissue culture dishes. A total of 1 mCi of lz51 and 12 lodobead catalysts (Pierce Chemical Co., Rockford, IL) were used per plate. Labeling was allowed to proceed at 0” for 30 min. To assay for the acquisition of temperature-de- pendent resistance to trypsin, cells that had been labeled with 1251 at 0” were washed five times with phos- phate-buffered saline (PBS) after removal of lodobead catalysts and placed at 37” in DMEM for 0, 30, or 60

min. After chase at 37”, the cells were placed into ice- cold DMEM containing 10 mM HEPES, pH 7.2, and 12.5 mg/ml trypsin (GIBCO BRL, Gaithersburg, MD) for 1 hr. At the end of this period, the trypsin was removed, and the cells were washed five times over 30 min with DMEM containing 10% fetal calf serum. Samples were lysed with the radioimmunoprecipitation assay (RIPA) buffer (Szurek et a/., 1990).

Analysis of gp70 in tissue culture medium

Clone 1 cells, psi-2 cells, or tsl-infected NIH 3T3 cells cultured at 34” were pulse radiolabeled for 30 min with [35S]methionine and [35S]cysteine and chased at 34” for 8 hr. After the chase, the medium was removed, and the cells were centrifuged at 3500 rpm for 15 min to pellet unattached cells and cell debris. The supernatant after this centrifugation was used directly for the immunoprecipitation and sodium dodecyl sul- fate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Szureketal., 1990). To determine the percentage of gp70 released into the culture medium, the amount of intracellular gp70 was also determined by lysing the cells, followed by immunoprecipitation with anti-gp70 and anti-p30 (to distinguish viral-specific protein expression in clone 1 and tsl-infected cells) and SDS-PAGE analysis (Szurek et a/., 1990). Quanti- tative measurements of radioactivities were carried out by the Bio Image System analysis (Bio Image Products, Ann Arbor, Ml).

Isolation of gp70 from the cell surface and post-ER intracellular compartment

Isolation of gp70 was carried out as described by Hare (1989) with the following modifications. Cells in 60-mm tissue culture dishes were labeled with both L-[35S]cysteine and L-[35S]methionine. Following various times of chase, the cells were kept at 4” for 10 min. Then 1.5 ml Dulbecco’s PBS (GIBCO) raised to pH 7.8 with NaOH and containing 200 pg/ml sulfosuccinimi- dyl-2-(biotinamido) ethyl-l ,3’-dithiopropriate (NHS-s-s- biotin) (Pierce Chemical Co.) was added to derivatize the cell surface proteins at 4”. The cells were gently shaken for 60 min. After lysis of cells, anti-gp70 antiserum (Szurek et a/., 1990) and fixed Staphylococcus aureus cells were sequentially added to lysates. Im- munoprecipitated proteins were solubilized in 50 ~12% SDS in 0.5 M Tris-HCI (pH 7.5) for 3 min at 95”. After brief centrifugation, the supernatants were mixed with 0.45 ml water and 25 ~1 streptavidin-agarose beads (Sigma Chemical Co., St. Louis, MO), and the suspen- sions were rotated end-over-end overnight at 4”. Beads were pelleted and the supernatants lyophilized. SDS-PAGE gel sample buffer (Szurek et al., 1990) was

TURNOVER AND DEGRADATION OF MURINE RETROVIRUS ENVELOPE PROTEINS 479

added to the samples, followed by a 3-min incubation at 95”. After centrifugation, the supernatants containing the envelope glycoproteins were analyzed by SDS- PAGE.

Treatment with L-methionine methyl ester

L-methionine methyl ester (20 mM, Sigma) containing medium (DMEM with 6% fetal calf serum and 4% new born calf serum) was used to replace the normal medium from the beginning of chase.

Metabolic labeling, radioimmunoprecipitation, SDS-PAGE, and autoradiography

Metabolic labeling, radioimmunoprecipitation, SDS- PAGE, and autoradiography were performed as described previously (Szurek et al., 1990). Goat antiser- ums prepared against Rauscher MuLV p30 (lot 77SOOOO87) and Rauscher MuLV gp69/71 (lot 79SOOO842) (Microbiological Associates, Inc., Be- thesda, MD) were used for the detection of the gag and envelope proteins, respectively. Goat antiserum against triton-disrupted MuLV (Microbiological Asso- ciates) was used to immunoprecipitate both gp70 and Prpl5E.

Quantitation of radioactivities of protein bands in SDS-PAGE

Radioactivities present in protein bands in all SDS- polyacrylamide gels in this studywere quantified by the Bio Image System.

RESULTS

Internalization and shedding of gp70 at the clone 1 cell surface

The possible fate of gp70 after it has reached the cell surface may include internalizing into intracellular compartment, shedding into extracellular medium, and/or being degraded at the cell surface. To determine whether internalization of gp70 occurs after it has reached the cell surface, a commonly used assay for internalization of the cell surface glycoprotein (Bretscher and Lutter, 1988; Lazarovits and Roth, 1988; Roth et a/., 1986; Watts, 1985) was performed. Clone 1 cells were incubated at 34” for 12 hr to allow synthesis and transport of the envelope protein to the cell surface. The surface of clone 1 cells were then labeled with 1251 at 0”. Cells were subsequently treated with trypsin in order to digest all cell surface gp70 proteins. Internalized gp70 should not be accessible to the protease. When trypsin was added to the cell culture medium at 0 time point, the labeled gp70 disap-

PP70-

1 2 3 4

FIG. 1. Internalization of gp70 at clone 1 cell surface. Clone 1 cells were iodinated for 30 min, and the amount of gp70 labeled at the cell surface was analyzed. Lane 1, cells without trypsin treatment after iodinization; lane 2, cells with immediate trypsin treatment after iodinization; lanes 3 and 4, cells treated with trypsin after 30 or 60 min incubation, respectively, at 37” (for experimental details, see Mate- rials and Methods).

peared (compare lanes 1 and 2 in Fig. l), indicating that no internalization had occurred. However, when labeled clone 1 cells were incubated at 37” for 30 or 60 min and subsequently treated with trypsin, a portion of ‘251-labeled gp70 became resistant to trypsin digestion (Fig. 1, lanes 3 and 4, respectively), suggesting that the internalization of gp70 at the clone 1 cell surface had occurred at these time points.

To examine whether shedding of gp70 occurred at the clone 1 cell surface, the cells were pulse radiolabeled with [35S]methionine and [35S]cysteine for 30 min at 34”. After an 8-hr chase at the same temperature, shedding of gp70 from clone 1 as well as from tsl-infected cells was analyzed as described under Mate- rials and Methods. Uninfected parental NIH 3T3 cells were used as control in the experiment. The result is presented in Fig. 2. Quantitative measurements of radioactivities by the Bio Image System indicated that less than 5% of the total gp70 (the intracellular gp70 plus the gp70 from the medium) was shed from clone 1 cells (compare lanes B2 and B4 in Fig. 2). The amount of gp70 shed by clone 1 cells was less than 8% of the total gp70 released by tsl-infected cells (compare lanes 62 and C2 in Fig. 2). Results presented in lanes 84 and C4 indicate that both clone 1 and tsl -infected cells retained similar amounts of intracellular gp70. Al- though the anti-gp70 antiserum also precipitated a protein with the molecular weight of 32 kDa from the cell lysates (see lanes A4, B4, and C4, as indicated by arrowhead), the same protein was not detectable in the culture mediums of all three different cell lines (see lanes A2, B2, and C2). Since no bands appear in lanes Al and A2, we conclude that none of the endogenous viruses in the parental (uninfected) NIH 3T3 cells (if they do exist) produce detectable amount of cross- reactive proteins in the culture medium.

480 YU AND WONG

A 6 c ‘1 2 3 4’ i23ii23i

,gPr130env

GP70 -Pr65gag

-pr40gag

-(P30

FIG. 2. Analysis of shedding of gp70 from clone 1 cells. This experiment was carried out as described under Materials and Methods, (A) The cell lysate and tissue culture medium from the untransfected and uninfected parental NIH 3T3 cells were used; (B) from clone 1 cells; (C) from &l-infected NIH 3T3 cells. Anti-p30 antiserum was used in lanes 1 and 3 and anti-gp70 antiserum was used in lanes 2 and 4. The samples in lanes 1 and 2 were from the tissue culture medium and in lanes 3 and 4 were from the cell lysates. Pr40Qag in lane C3 is an intermediate cleavage product of Pr65Qw (Soong et al., 1984; Dickson et a/., 1984). Arrow indicates the BiP protein determined in a previous study (Szurek et al., 1990); arrowhead desig- nated an unidentified protein (lanes A4, B4, and C4) which has an M, of 32,000.

Synchronization of transport of gp70 and regulation of amount of gp70 in post-ER cellular compartments

To study comparative turnover of gp70 in the post- ER intracellular compartment and at the cell surface, it is important to determine whether [35S]-labeled gPr80”“” continues to be processed into gp70 at the late stage of chase. Since there is no conclusive evidence that all gp70 will arrive at the cell surface, contin- uous transport of gPr80e”“from the ER to the Golgi to be processed into gp70 at the late stage of chase may increase the amount of the intracellular gp70. This, in turn, might cause the overestimation of the accumulation rate of internalized cell surface gp70 and therefore may complicate interpretation of the data. We, therefore, examined whether gPr80e”” in clone 1 cells was processed into gp70 at the later stage of chase at 34 and at 39”. Two plates of clone 1 cells were pulsed and chased at 34”, and the cells in plate 1 were lysed after a 4-hr chase. At the same time, the cells in plate 2 were transferred to 20” and chased for another 3 hr [according to the studies in other laboratories, the degradation of membrane protein is inhibited at 20” (Hare, 1988; Marsh et a/., 1983), but membrane glycoproteins at the ER still can transport to the cis and the medial Golgi (Saraste and Kuismanen, 1984) although at reduced levels]. The results shown in Fig. 3A indicate that after a 4 hr-chase, gPr80e”” was still processed into gp70 at the permissive temperature (compare lanes 1 and 2). However, no cleavage of gPr80”“” was detected when

the chase was carried out at 39.5” (compare lanes 1 and 2, Fig. 3B). These results indicate that at the restrictive temperature, over 99% of Tsl gPr80”“’ was incompetent for transport from the ER to the Golgi. On the other hand, when clone 1 cells were incubated at 34” and then shifted to 39.5”, only a portion of gPr80e”” exited from the ER and entered the Golgi where it was processed to the gp70 and Prpl5E. The amount of gPr80e”’ delivered to the Golgi compartment was directly proportional to the time of incubation at the permissive temperature (Vu, 1992). Therefore, temperature-shift experiments can be utilized to regulate the transport of labeled gPr80e”” from the ER to the Golgi, and the amount of the gp70 entering the post-ER intracellular compartment can also be blocked at the late stage of chase by shifting the temperature to 39.5”.

Studies of turnover of gp70 in the post-ER intracellular compartment and cell surface

Based upon the above results, temperature-shift experiments were designed to analyze the distribution of gp70 between the cell surface and the post-ER intracellular compartment and the kinetics of transfer of gp70 between these two compartments. To isolate gp70 at the cell surface in this experiment, a recently described biotinylation/recovery assay of cell surface proteins (Hare and Lee, 1989; Hare, 1989; Hare, 1990; Hare and Taylor, 199 1) was utilized. In this experiment, the clone 1 cells were radiolabeled at 39.5”, then chased at the same temperature for 30 min. After that, the cells were held at 34” for 40 min before returning them to 39.5” for the remaining period of the chase.

A

gPr80e”V-

!3P70-

t 2

B

gPr80env- gp70-

FIG. 3. Pulse-chase analysis of gPr80”” processing. (A) The clone 1 cells were pulse radiolabeled for 10 min with [%]methionine and [%]cysteine and chased at 34” for 4 hr (lane 1); after this initial chase, clone 1 cells were transferred to 20” and chase was contin- ued for another 3 hr (lane 2). (B) The [35SJ-radiolabeled clone 1 cells were chased at 34” for 4 hr (lane 1) at 39.5” for 4 hr (lane 2).


After the chase, the cells were derivatized at 4” with 200 pg/ml membrane-impermeant NHS-s-s-biotin for 1 hr to assure saturation of derivatization sites. Separa- tion of derivatized and underivatized gp70 from im- munoprecipitates by selective binding to streptavidin- agarose beads were conducted as described under Materials and Methods to isolate the gp70 in the post- ER intracellular compartment and at the cell surface. The results from Fig. 4 show that although there was shedding of gp70 from cell surface, the turnover rate of MoMuLV gp70 at the cell surface was reproducibly similar (in five separate experiments) to that of the post-ER intracellular MoMuLV gp70.

To interpret the data in Fig. 4 correctly, it was, in our experiment, a prerequisite that NHS-s-s-biotin-derivatized gp70 does not contain a large amount of intracellular gp70. In order to examine whether NHS-s-s-biotin can access the proteins in the cytoplasmic side of the cell plasma membrane, MoMuLV ts3 virus-infected NIH 3T3 cells were used. At the restrictive temperature, unlike wild-type MoMuLV, the Pr65g”g precursor protein of ts3 accumulated at the cytoplasmic side of cell plasma membranes and was not released into the medium as virions (Witte and Baltimore, 1978; Wong and MacLeod, 1975; Yuen and Wong, 1977). On the other hand, gp70 in ts3-infected cells at 39” behaves

gPr80env

QP70

Chasethr) 2 4 8 Q 2 4 8 9

C

“2 -I ,,.I. I .,.,.,.I. I

3 4 5 8 7 8 9 10

Time.hr

FIG. 4. Turnover rate in the post-ER intracellular compartment and cell surface of clone 1 cells. Clone 1 cells were pulse radiolabeled with [35S]methionine and [35S]cysteine for 30 min at 39.5”, followed by a 30.min chase at 39.5”, then 40 min at 34”. After that, the cells were chased at 39.5” for the remaining times as indicated: (A) intracellular envelope proteins; (B) gp70 at the cell surface; (C) quantitation of radioactivity present in gp70 bands from (A) and (B).

* QP70-

Pr859W-

123458

B

;Pr80env

-QP70 -Pr85QaQ

1 2

FIG. 5. Comparison of amounts of Pr65gag and gp70 in the rntracel- lular compartment and at the cell surface using the biotinylation/re- cover-y assay. MoMuLV t.s3-infected NIH 3T3 cells were pulse radiolabeled with [35S]methionine and [35S]cysteine for 30 min at 39”, followed by a 2.5-hr chase at 39”. After the chase, the cells were derivatized at 4” with NHS-s-s-biotin for 1 hr. Half of the cell lysate from each dish was immunoprecipitated with anti-p3WW antiserum [(A) lanes 1-3, (B) lane 11; the other half was immunoprecipitated with anti-gp70 antiserum [(A) lanes 4-6, (B) lane 21; immunoprecipitated proteins were then mixed with streptavidrn-agarose beads as described under Materials and Methods. Beads were pelleted, while the supernatants were transferred to another set of tubes and lyophilized. SDS-PAGE sample buffer (Szurek er a/., 1990) was added to the tubes containing either beads or the lyophilized samples, followed by a 3-min incubation at 95”. After centrifugation the supernatants were analyzed by SDS-PAGE. (A) The samples were from the precipitated streptavidin-agarose beads. Arrow indicates the position of gPr80e”’ normally detected when intracellar envelope proteins from MoMuLV-infected cells were analyzed. (B) The samples were from the lyophilized supernatants. Arrowhead designates the BiP protein determined in a previous study (Szurek et a/., 1990).

the same as wild-type gp70 and transports to the cell surface. The results in Fig. 5 show that NHS-s-s-biotin detected less than 1 YO of intracellular Pr65gag, whereas about 40% total gp70 was labeled, suggesting that the NHS-s-s-biotin-derivatized gp70 is primarily from the cell surface. Also, no gPr80e”” was detected at the cell surface, which is consistent with a previous report that NHS-s-s-biotin only accesses the cell surface membrane proteins (Hare, 1989).

Another experiment was conducted to examine the effect of the cell surface gp70 isolation with the in- creased amount of NHS-s-s-biotin and streptavidin- agarose in the biotinylation/recovery assay. We found that a twofold increase of NHS-s-s-biotin and streptavidin-agarose does not increase the amount of gp70 isolated from the clone 1 cell surface when compared with the standard condition, described under Materials and Methods (Vu, 1992). This suggests that the con- centration and volume of NHS-s-s-biotin solution used

482 YU AND WONG

- gP70

--a-- PrplSE

- gpi’a(MME)

- -.- - Prpl SE(MME)

OI,.,,.,.,,,,.,,..,,,,~I 3 13 23

The (hr)

FIG. 6. The turnover rates of gp70 and Prpl5E with or without the addition of L-methionine methyl ester in the temperature-shift experiments. The clone 1 cells were radiolabeled at 39.5” for 30 min, then chased at the same temperature for 30 min. After that, the cells were held at 34” for 20 min before returning them to 39.5” to continue the chase. The rest of the experimental procedures are as described under Results. Radioactivity present in gp70 and Prpl5E bands in SDS-PAGE were quantitated by the Sio Image System. Each point represents an average of radioactivities from three experiments. Error bars represent one standard error of the mean.

in the above study was optimum to react to gp70 at the cell surface. Therefore, the gp70 isolated using the standard method described above should be able to represent the amount of gp70 in the cell surface of the clone 1 cells.

Turnover of gp70 and Prpl5E of clone 1 cells cultured in the presence of L-methionine methyl ester

Although L-methionine methyl ester does not prevent the intracellular transport of envelope protein (Willey et al., 1988) as a lysosomotropic agent, it can reduce the intracellular degradation by inhibiting membrane fusion between endocytic vesicles and lysosomes (Matrisian et al., 1987; Merion and Sly, 1983; Hare, 1988). It also raises the lysosome pH (Poole and Ohkuma, 1981). To follow the turnover of a small but finite amount of gp70 and Prpl5E in the presence of L-methionine methyl ester in the culture medium or in the normal DMEM medium, the clone 1 cells were radiolabeled at 39.5” for 30 min, then chased at the same temperature for 30 min. After that, the cells were held at 34” for 20 min before returning them to 39.5’ to continue the chase. L-methionine methyl ester was added to the medium throughout the chase period. The result of this experiment is shown in Fig. 6. Surpris- ingly, it was found that Prpl5E was degraded faster than gp70 in the presence of L-methionine methyl ester in the tissue culture medium, whereas similar turnover

rates were observed if the labeled cells were chased in the normal DMEM medium.

DISCUSSION

The results reported here present several new find- ings about the turnover of retroviral envelope proteins. By taking advantage of the temperature-sensitive phenotype of tsl envelope proteins in clone 1 cells, we were able to synchronize the entry of a limited amount of gp70 into the post-ER intracellular compartments and follow the subsequent fate of the proteins. We found that gp70 in the intracellular compartment and gp70 at the cell surface have a similar turnover rate. These data are different from those reported previously by Hare (1989) who showed that the env proteins of MoMuLV in psi-2 cell surface turned over more rapidly than the intracellular pool of these proteins. Based on the observation that the decrease of radiolabeled gp70 at the cell surface coincided with an increase of radiolabeled intracellular gp70 during a 6-hr chase, Hare suggested that the surface envelope proteins internalized and accumulated in a compartment between cell surface and proteolytic compartment(s) before they fi- nally transferred to proteolytic compartment(s). This ar- gument depends, however, on the hypothesis that all cell surface gp70 internalized and that no external shedding or release took place. This is not the case. Although Hare was not able to detect gp70 in the tissue culture medium of psi-2 cells (Hare, 1989), other researchers using the same cells reported a large amount of gp70 (Jones et a/., 1990) released into the medium either as free molecules or as components of noninfectious virions. We also found that at least 50% of the total gp7Os (i.e., the intracellular gp70 plus the gp70 from the medium) from psi-2 cells is released into the medium after an 8-hr chase at 34”; most of the released gp7Os are as components of virions (Vu and Wong, unpublished data). This finding complicates the interpretation of the psi-2 cell study. Thus, the faster turnover rate of the envelope proteins from the surface of psi-2 cells relative to those located intracellularly could be interpreted as a combined effect of shedding and internalization of the former. On the other hand, when clone 1 cells were used in our study, we found that less than 5% of the total gp70 from clone 1 cells was detectable in the medium after an 8-hr chase at 34”. Our results suggest that the internalized cell surface gp70 proteins do not accumulate in the intracellular compartment(s). This indicates that the cell is able to rapidly establish and maintain an equilibrium of envelope protein distribution between the cell surface and intracellular compartment(s) probably due to the combined effects of internalization, recycling to the cell


surface, and transferring to the degradation compartment(s).

Nevertheless, the concept of long-term intracellular accumulation of the internalized surface protein, which Hare proposed in his study on gp70 (Hare, 1989) is an interesting one because it is relevant to the mecha- nism for establishing intracellular pools of the cell plasma membrane protein. According to the concept, a specific cell surface membrane protein may transfer between the cell surface and intracellular pools by one of two different kinetics. Some membrane proteins may achieve equilibrium distribution between the cell surface and intracellular pools soon after they reach the cell surface, such as gp70 presented in this study and the low-density lipoprotein receptor in skin fibroblasts reported by Hare (1990). Other proteins may also internalize after they reach the cell surface, but those internalized proteins may continue to accumulate in the intracellular pool for a period of time before redistributing to the other intracellular compartment(s). Such an accumulation remains to be demonstrated.

It is interesting to note in this study that shedding only occured to a very small percentage of gp70 from clone 1 cells, which is in contrast to the relatively high level (over 50%) of shedding of gp120 that was reported for cells expressing the env gene of HIV alone (Chakrabarti et a/., 1990; Earl et al., 1991; Kieny et a/., 1986). The hydrophobic interactions between the extracellular domain (SU) and the transmembrane domain (TM) of the retroviral envelope protein apparently enhance the formation of the heterodimer between gpl20 and gp41 of HIV and the heterodimer between gp70 and Prpl5E of MoMuLV. However, it might be the disulfide bonds between the two subunits (SU and TM) of the envelope proteins that ultimately determine the stability of the envelope heterodimer. The small amounts of gp70 shedding from clone 1 cells reported here may be due to two disulfide bonds existing between gp70 and Prpl5E (Pinter and Fleissner, 1977; Pinteretal., 1978) whereas the large amount of gpl20 shedding in the case of HIV may be due to the absence of the disulfide bond between the gp120 and gp41 heterodimer, which renders the gpl20 less stable (Ko- walski eta/., 1987). At present, the percentage of gp70 directly shed from the virion is not known. If the association between gp70 and Prpl5E in the virion is similar to that at the cell surface of clone 1 cells, there will be a lot less free gp70 in MoMuLV-infected mouse than free gpl20 in HIV-infected patients, assuming that the num- ber of viral particles in the bloodstreams of both sub- jects are the same. Because the extracellular domain of the envelope protein alone has been suggested to be associated with the cytopathic effect in target cells (Brenneman et a/., 1988; Gliniak and Kabat, 1989; Ra-

sheed et a/., 1986) the viruses with different envelope shedding abilities may have different pathogenic po- tentials and utilize different molecular mechanisms of pathogenesis. This possibility can be examined in part by removing the disulfide bonds between gp70 and Prpl5E by the site-directed mutagenesis of the tsl viral genome.

The extracellular domain of plasma membrane proteins, like gp70, if not shed into the medium, is degraded in lysosomes. Evidence for lysosomal involve- ment in plasma membrane degradation includes studies on inhibitors (Christopher and Morgan, 1981; Stoscheck and Carpenter, 1984) and morphological studies (Beguinot et al., 1984, 1986; Dunn et al., 1986) as well as studies utilizing in vitro digestions (Schneider et al., 1981) and study of the biochemical location of membrane degradation products (Hare and Huston, 1985; Russel and Mayer, 1983). However, the fate of the cytoplasmic and transmembrane domains of membrane proteins has yet to be determined. After internalization takes place, the membrane-containing envelope proteins may invaginate and form multivesi- cle bodies, where the cytoplasmic and transmembrane domains may be digested (Biberfeld, 1971). Alter- nately, the cytoplasmic domain of the envelope protein may be released into the cytosol and degraded there. Our results presented in this report show that, in the presence of L-methionine methyl ester, the turnover rate of Prpl5E is much faster than that of gp70, suggesting that there may be two different enzyme systems involved in the proteolytic degradation of gp70 and Prpl5E.

Since L-methionine methyl ester inhibits proteolytic degradation in lysosomes or endosomes mainly by in- creasing the pH inside these compartments (Poole and Ohkuma, 1981), gp70, being more stable in the presence of L-methionine methyl ester is most likely degraded inside the lysosome. On the other hand, the faster turnover rate of Prpl5E in the presence of L-

methionine methyl ester suggests that it may not be degraded inside the lysosome. It is also possible that gp70 and Prpl5E are degraded by the same cellular proteolytic system but that the rates of degradation are different between gp70 and Prpl5E in the presence of L-methionine methyl ester. Whetherthe enhanced turnover rate of Prpl5E is due to its cytoplasmic domain is not clear. Similar studies on cells expressing a mutant envelope protein alone with a deleted cytoplasmic domain should provide the answer to this question.

Among the best-characterized membrane glycoproteins, the envelope protein of MoMuLV has its special structural feature: the precursor envelope protein, gPr80e”“, which is synthesized as a monomeric protein in the ER before trimerization takes place, was pro-

484 YU AND WONG

cessed into gp70 and Prpl5E in the Golgi complex. gp70 and Prpl5E are tightly associated as a heterodimer. Studying the events related to these two subunits in subcellular compartmentation and turnover may yield useful information about the extracellular domain and the transmembrane and cytoplasmic domain of plasma membrane proteins in general.

ACKNOWLEDGMENTS

We thank Drs. Dennis T. Brown and M. E. Reichmann for their critical reviews of this manuscript. We also wish to thank C. McKin- ley for her assistance in the preparation of this manuscript. This investigation was supported by Public Health Service Grant CA 45124 from the National Cancer Institute and Al 28283 from the National Institute of Allergy and Infectious Diseases.

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Studies on compartmentation and turnover of murine retrovirus envelope proteins

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