THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 17, Issue of September 10, pp. 6955-6962, 1975

Printed in U.S.A.

Site of Synthesis of Membrane and Nonmembrane Proteins of Vesicular Stomatitis Virus*

(Received for publication, January 27, 1975)

TRUDY G. MORRISON+

From the Department of Microbiology, University of Massachusetts Medical School,

Worcester, Massachusetts 01650

HARVEY F. LODISH§

From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Upon infection of Chinese hamster ovary cells (CHO), vesicular stomatitis (VSV) virus synthesizes two membrane proteins (the VSV glycoprotein and the VSV matrix or membrane (M) protein) and three nonmembrane proteins (the VSV nucleocapsid, the viral transcriptase, and an NS protein). We have used the VSV-infected cell as a model system for the study of the site of synthesis of these membrane and nonmembrane proteins. We have isolated VSV mRNA from free polyribosomes, membrane-bound polyribosomes, and the postribosomal supernatant, and identified the individual species of VSV mRNA present in each fraction. The mRNA which encodes the VSV glycoprotein is found exclusively on mem- brane-bound polyribosomes, while the mRNAs which encode the VSV, M, N, and NS proteins are found in free polyribosomes, in the membrane fraction of the cell, and in the postribosomal supernatant. Our results suggest that the VSV glycoprotein is synthesized exclusively on membrane polyribosomes, while at least some of the M, N, and NS proteins are made on free polyribosomes.

Polyribosomes of eukaryotic cells are both free in the cytoplasm and associated with the membranous fractions of the cell (1, 2). Extensive evidence indicates that membrane- bound polyribosomes are engaged in the synthesis of proteins which are transported from the cell, while free polyribosomes are engaged in the synthesis of proteins which remain in the cell sap (3-6). For example, membrane-bound polyribosomes are the site of synthesis of immunoglobins (7), and albumin (3-5), proteins which are secreted from the cell, while free polyribosomes preferentially synthesize ferritin (3-5), a protein which remains in the cell.

The site of synthesis of membrane proteins is, however, less clearly defined. Theories of membrane biogenesis include the idea that plasma membrane is derived from endoplasmic reticulum which has been processed through the Golgi ap- paratus (8). Thus, surface membrane proteins might be con- sidered a special class of secreted proteins. However, Lodish and Small (9, 10) and Lowe and Hallinan (11) have described synthesis of membrane proteins on free polyribosomes.

*This work was supported in part by American Cancer Society Grant VC-167 and by a General Research Science grant awarded to the University of Massachusetts, and by Grants AI-08814 and AM-15929 from the National Institutes of Health.

i Fellow of the Damon Runvon Memorial Fund for Cancer Research. 5 Recipient of National Institutes of Health Research Career Devel-

opment Award GM 50175.

Cells infected with vesicular stomatitis virus (VSV) 1 provide an ideal system to study the site of synthesis of membrane and nonmembrane proteins. The genome of VSV is a single RNA molecule (molecular weight = 4 x 106) which is transcribed by a virion-associated polymerase (12) into complementary mRNA molecules (13, 14). The virus replicates exclusively in the cytoplasm (15). In the infected cell, both parental and progeny negative strands are transcribed to produce five monocistronic messenger RNAs (14, 16). Two of these mRNAs encode two membrane proteins, the viral glycoprotein (G) and the viral M protein (matrix or membrane protein). The glyco- protein is found on the external surface of the plasma mem- brane and, following budding of the virus from the cell, becomes the spike of the mature virion (17-20). The M protein also becomes associated with the host cell membrane, although little of this protein protrudes to the outer surface of the membrane (21). M protein is believed to line the inner surface of the bilayer, and to connect with the inner nucleocapsid core (19, 20). The three core proteins, which may be considered nonmembrane proteins, are the nucleocapsid (N) protein, the L protein (viral transcriptase) (22), and the NS protein.

When separated on sucrose gradients, the five VSV mRNAs

‘The abbreviations used are: VSV, vesicular stomatitis virus; CHO cells, Chinese hamster ovary cells; Hepes, N-2-hydroxyethylpipera- zinc-N’-2.ethanesulfonic acid.

6955

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from

6956

are resolved into two size classes, a homogeneous species sedimenting at 28 S, and a heterogeneous group of RNAs sedimenting between 13 S and 15 S (11, 23). We have previously shown that the VSV 28 S RNA, when added to a cell-free extract from rabbit reticulocytes, directs the synthesis of a polypeptide the size of the VSV L protein, while the VSV 13 to 15 S RNAs direct the synthesis of the VSV M, N, NS

proteins, and probably the VSV G protein (16,24, 25). In this paper, we have investigated the cellular localization

of the VSV 13 to 15 S RNA. We have isolated VSV mRNA from membrane and nonmembrane fractions of the infected cell. We have synthesized the VSV glycoprotein in a cell-free system, and we show that the VSV mRNA which encodes the VSV glycoprotein is found exclusively in the membrane fraction of the infected cell, while mRNA which encodes the VSV mem- brane (M) protein is found largely on free polyribosomes and in the postribosomal supernatant, but an appreciable amount is in the membrane fraction. We have also found the mRNAs which encode the VSV N and NS proteins on free and membrane-associated polyribosomes as well as the postriboso- ma1 supernatant. Our results suggest that G protein is made exclusively on membrane polyribosomes, while at least some M (and N and NS) proteins are made on free polyribosomes.

MATERIALS AND METHODS

Purification of VSV mRNA from Cell Fractions

Method A: Polyribosome Gradients-Chinese hamster ovary- (CHO) cells growing at 37” were infected with VSV at a multiplicity of 3 plaque-forming units/cell as described by Huang et al. (13). except that 5 fig/ml of actinomycin D were added at the beginning of infection. [3H]Uridine (70 Ci/mmol, New England Nuclear Corp.; 5 pCi/ml) was added 2 hours postinfection. Infected cells were harvested at 4 to 5 hours postinfection, resuspended in sucrose-Buffer A (0.05 M Tris, pH 7.5; 0.025 M KCl; 0.005 M Mg acetate; 0.25 M sucrose) and disrupted with seven strokes of a tight fitting Dounce homogenizer. The nuclei were removed by centrifugation (1000 x g for 2 min), and the cytoplasmic extract was layered onto a 5 to 30% sucrose gradient buffered with Buffer A or Buffer B (0.01 M Tris, pH 7.5; 0.5 M NaCl; 0.05 M MgCl,). The gradients were centrifuged at 4” for 2.25 hours at 26,000 rpm in a Beckman SW 27 rotor. Fractions were collected through a Gilford recording spectrophotometer. The polyribosome region (bottom of the gradient to the 80 S ribosomes) of the gradient was pooled. The RNA was precipitated with ethanol and resuspended in sodium dodecyl sulfate buffer (0.5% sodium dodecyl sulfate; 0.01 M

Tris, pH 7.4; 0.1 M NaCl; and 0.001 M EDTA). This material is defined as VSV RNA which was associated with free polyribosomes. The material at the top of the gradient, material with a sedimentation value of less than 40 S, was pooled, and the RNA was precipitated with ethanol and resuspended in sodium dodecyl sulfate buffer. This mater- rial is defined as VSV mRNA which was not associated with poly- ribosomes in the infected cell, and is referred to as the postribosomal supernatant fraction. Finally, the pellet of the polyribosome gradient was resuspended in sodium dodecyl sulfate buffer. This material has been defined as the membrane fraction of cells (2. 26). The solubilized RNAs were isolated from free polyribosomes, membranes, and the postribosomal supernatant, layered onto 15 to 30% sucrose gradients buffered with sodium dodecyl sulfate buffer, and centrifuged at 20” at 24,000 rpm for 17 hours in a Beckman SW 27 rotor. The gradient frac- tions containing 13 to 15 S RNA were pooled. The RNA was precipitated twice with ethanol, and finally dissolved in water.

Method B: Discontinuous Sucrose Gradients-Cytoplasmic extracts of VSV-infected CHO cells were prepared as described under “Method A,” with the exception that polyvinyl sulfate (50 fig/ml) was added to the cytoplasmic extracts to inhibit degradation of the RNA. The extracts were then made 30% with respect to sucrose, and layered on top of preformed discontinuous sucrose gradients, as described by Klerik et al. (27). The discontinuous gradients, made in 38-ml centrifuge tubes, contained 10 ml of 60% sucrose (dissolved in 0.001 M

Tris, pH 7.4; 0.001 M MgCl,), 10 ml of 45% sucrose, and 10 ml of 40% sucrose. Five milliliters of cytoplasmic extract were layered on top of

the 40% sucrose. The tubes were filled to the top with 25% sucrose. The gradients were centrifuged for 16 hours at 4” at 23,000 rpm in a Beckman SW 27 rotor. Fractions were collected through a Gilford recording spectrophotometer. Small aliquots of each fraction of the gradient were removed, precipitated with trichloroacetic acid, and the radioactivity present in each precipitate determined by liquid scintillation counting (see Fig. 6). There was a major peak of radio- activity at the interface between the 60% sucrose and the 45% sucrose layers. This peak was defined as membrane Band 1. There was also a minor peak of radioactivity at the interface between the 40% and 45% sucrose layer. This peak was defined as membrane Band 2. The material in the 30% sucrose layer (location of the cytoplasmic ex- tract prior to centrifugation) and at the interface between the 30% and 40% sucrose layers was defined as VSV RNA which was not associated with polyribosomes in the infected cell and is referred to as the supernatant fraction. The pellet of the discontinuous sucrose gradient was defined as the free or nonmembrane-associated poly- ribosomes (27).

The RNA in each fraction was precipitated with ethanol and resuspended in sodium dodecyl sulfate buffer. The solubilized RNA from the four cell fractions was layered onto 15 to 30% sucrose gradients buffered with sodium dodecyl sulfate buffer and centrifuged at 20” at 24,000 rpm for 17 hours in a Beckman SW 27 rotor. Gradient fractions containing 13 to 15 S RNA were pooled. The RNA was precipitated twice with ethanol, and finally dissolved in water.

Cell-free Protein Synthesis

Cell extracts used to direct cell-free protein synthesis were derived from wheat germ and prepared as described by Roberts and Paterson (28). Reactions (50 ~1) contained, per ml, 20 pmol of Hepes, pH 7.6, 2 wmol of dithiothreitol, 1 rmol of ATP, 0.2 pmol of GTP, 8 pmol of creatine phosphate, 40 pg of creatine phosphokinase, 0.020 to 0.030 pmol of 19 unlabeled amino acids, 76 hmol of KCl, 3 pmol of magnesium acetate, 0.20 ml of wheat germ extract, and 250 KCi of [3”S]methionine (120 Ci/mmol, New England Nuclear Corp.). Incuba- tion was for 120 min at 22’.

Polyactylamide Gel Electrophoresis Proteins

Cell-free reactions were terminated by the addition of ribonuclease A (50 pg/ml) and incubated for 10 min further. The products of the reaction were resolved on 10% polyacrylamide gels (6 mm x 15 cm) containing 0.1% sodium dodecyl sulfate and 8 M urea as described previously (29). The gels were prepared for radioautography and the resulting radioautograms scanned with a Joyce-Loebel microdensi- tometer as described by Lodish and Desalu (30).

Formamide-Polyacrylamide Gel Electrophoresis-RNA

The formamide-polyacrylamide gels used here to resolve VSV 13 to 15 S RNA are as described by Duesberg and Vogt (31), except that slab gels run in an E-C slab gel apparatus were used (32). The gel was subjected to electrophoresis at 80 volts for 16 hours. Each channel in the slab was then excised, frozen in an acetone-Dry Ice solution, and sliced horizontally with a razor blade into slices 1 mm thick. Each slice was placed in 7 ml of toluene which contained 0.4% Omnifluor (New England Nuclear Corp.) and 3.5% Protosol (New England Nuclear Corp.) or 3.5% NCS (Amersham/Searle). The vials were incubated overnight with shaking at 37”, cooled, and the radioactivity in each was determined by liquid scintillation counting.

Marker VSV Cytoplasmic Proteins

CHO cells infected with VSV at a multiplicity of three in the presence of actinomycin D (5 pg/ml) were labeled with [Yjlmethionine (120 Ci/mmol, New England Nuclear Corp.) between 4 and 4.5 hours postinfection. The cells were harvested and disrupted in 1% NP40 (Shell Oil Co.). The nuclei were removed by centrifugation.

Tryptic Peptide Analysis

VSV proteins (both cytoplasmic protein and proteins made in cell- free reactions) were resolved on 10% polyacrylamide gels. The location of each protein within the gel was determined by radioautography of the fixed, dried gel.

Trypsin digestion of the individual VSV proteins was accomplished without prior elution of the polypeptide from the gel slice. We found that trypsin is able to digest polypeptides that are still within the gel matrix. The resulting tryptic peptides elute from the gel and are

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from

6957

recovered in the surrounding solution. The region of the gel containing each individual protein was excised and placed in 1 ml of a 1% ammonium bicarbonate solution which contained 100 pg of trypsin. The solution was incubated at 37” with shaking overnight. The gel slice was placed in fresh trypsin-ammonium bicarbonate solution and incubation was continued for another 4 hours. The trypsin-ammonium bicarbonate solutions were pooled and lyophilized twice. We recovered from 60 to 80% of the radioactivity from the gel.

The [35S]methionine tryptic peptides were analyzed by paper ionophoresis at pH 3.5 as described previously (33, 34).

RESULTS

Translation of VSV 13 to 15 S RNA Isolated from the Membrane Fraction of Infected Cells-VSV-infected cells were labeled with [3H]uridine in the presence of actinomycin D, so that only VSV-specific RNA was labeled. Cytoplasmic extracts of VSV-infected cells were prepared and a high speed particu- late fraction of the infected cell extracts was isolated as described in “Method A” under “Materials and Methods.” This material has been defined as the membrane fraction of eukaryotic cells (2, 26). VSV 13 to 15 S RNA isolated from the membrane fraction was added to a cell-free system derived from wheat germ. After incubation at 22” for 120 min, the reaction products (radioactively labeled with [YS]methionine) were analyzed by electrophoresis on 10% polyacrylamide gels. Microdensitometer scans of autoradiographs of the fixed, dried gels are presented in Fig. 1. Marker proteins also radioac- tively labeled with [35S]methionine were subjected to electro- phoresis in parallel polyacrylamide gels.

Just as had been previously found using RNA isolated from the polyribosomes of VSV-infected cells disrupted with the detergent NP40 (16), membrane-associated RNA stimulated the synthesis of polypeptides approximately the size of the VSV nucleocapsid (N), the matrix or membrane protein (M), and the NS protein. In addition, VSV 13 -to 15 S RNAs derived from the membrane fraction of infected cells also directed the synthesis of a significant amount of a polypeptide which migrated on polyacrylamide gels slightly faster than the authentic VSV glycoprotein (G).

Tryptic Peptide Analysis of the VSV Glycoprotein-Because of the carbohydrate residues, glycosylated polypeptides mi- grate on sodium dodecyl sulfate polyacrylamide gels slower than would be predicted by the molecular weight of their protein moieties (35). It seemed likely that the polypeptide which migrated slightly faster on polyacrylamide gels than the authentic VSV glycoprotein was VSV glycoprotein. Proof that this new polypeptide made in the cell-free system was VSV glycoprotein required tryptic peptide analysis of the cell-free product and the authentic VSV glycoprotein.

Cytoplasmic VSV proteins and VSV proteins made in the cell-free reaction directed by membrane-associated VSV 13 to 15 S RNA were subjected to electrophoresis on parallel polyacrylamide gels. The regions of the polyacrylamide gels which contained glycoprotein were excised and digested with trypsin. The resulting tryptic peptides were resolved by paper ionophoresis at pH 3.5. The [35S]methionine-labeled tryptic peptide patterns obtained from the authentic VSV glycopro- tein and the presumptive G protein made in the cell-free reaction are identical (Fig. 2). Thus, the VSV 13 to 15 S RNAs

, derived from the membranes of VSV-infected cells contained messenger RNA which can direct the cell-free synthesis of the VSV glycoprotein. That the glycoprotein made in the cell-free system migrated faster on polyacrylamide gels than the au- thentic viral glycoprotein may indicate that the protein made

NS N G

extract PIUS VSV 13s RNA

--_I_-.- I- - -I I 9 8 7 6

(bottom) cm

d 5 (top)

FIG. 1. Microdensitometer tracing of autoradiographs of polyacryl- amide gel analysis of VSV proteins. Top, authentic viral proteins made in VSV-infected CHO cells. Bottom, VSV proteins made in a cell-free extract of wheat germ directed by VSV 13 to 15 S RNA isolated from the membrane fraction of the VSV-infected cell. The reaction which (50 ~1) contained 10 PCi of [SsS]methionine was incubated for 120 min at 22”. The reaction was stopped with ribonuclease A. As calculated from a 5-~1 aliquot, the total incorporation of radioactivity into protein was 260,537 cpm. Reactions containing no added RNA contained 11,340 cpm trichloroacetic acid-precipitable material.

Cell Free Product

CM

FIG. 2. Paper ionophoresis of [35S]methionine-labeled tryptic pep- tides derived from the VSV glycoprotein. Bottom, tryptic peptides derived from authentic VSV glycoprotein. Top, tryptic peptides derived from VSV glycoprotein made in a cell-free reaction. Wheat germ cell-free reactions directed by VSV 13 to 15 S RNA associated with the membrane fraction of the infected cell were subjected t.o polyacrylamide gel electrophoresis as shown in Fig. 1. The presumed glycoprotein was digested with trypsin as outlined under “Materials and Methods.” Tryptic peptides of the authentic VSV glycoprotein were obtained from polyacrylamide gels containing VSV proteins made in VSV-infected cells. The tryptic peptides were subjected to paper ionophoresis at pH 3.5, 40 volts/cm, for 3 hours.

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from

6958

in the cell-free system is unglycosylated or partially glycosyl- ated.

Tryptic Peptide Analysis of the VSV N, NS, and M Proteins-Roberts et al. (36) have reported that translation of exogenous messenger RNA in wheat germ extracts often results in premature termination. A single mRNA often directs the synthesis of several polypeptides of various sizes, all of which contain the same NH, terminus’and which have related tryptic peptide patterns. We have also seen this phenomenon.2 There- fore, to show conclusively that VSV 13 to 15 S RNA derived from membranes does, in fact, direct the synthesis of four different and authentic VSV proteins, tryptic peptide patterns obtained from each of the cell-free products were compared to those derived from authentic VSV M, N, and NS proteins.

The products of cell-free reactions directed by VSV mem- brane-associated RNA as well as authentic VSV proteins present in cytoplasmic extracts of VSV-infected cells were subjected to polyacrylamide gel electrophoresis on parallel gels. Gel slices containing each polypeptide were digested with trypsin, and the resulting tryptic peptides resolved by paper ionophoresis at pH 3.5. Fig. 3 shows the pattern of tryptic peptides obtained from each polypeptide. The tryptic peptide patterns of the cell-free products are similar to those obtained from the corresponding cytoplasmic marker proteins. Thus, VSV 13 to 15 S RNA associated with membranes of infected cells contain messenger RNA for four different VSV proteins.

Distribution of VSV 13 to 15 S RNA in Various Cell Fractions-The fact that mRNAs, which encode both VSV membrane and nonmembrane proteins, are present in the membrane fraction of infected cells led us to explore the localization of VSV 13 to 15 S mRNA in various cell fractions.

VSV-infected cells were labeled with [3H]uridine and har- vested between 4.25 and 5.5 hours after infection. The cells were disrupted with a Dounce homogenizer, and the nuclei were removed by centrifugation. The resulting cytoplasmic extract was analyzed on 5 to 30% sucrose gradients. Following centrifu- gation, the gradients were divided into three fractions-the pellet (membranes), the free polyribosomes (bottom of the gradient to the 80 S region), and the fractions above the 40 S ribosomal subunit (postribosomal supernatant) (see under “Method A”).

VSV RNA was isolated from each fraction as described under “Materials and Methods,” and the amount of $H- labeled 13 to 15 S RNA in each was determined by sucrose gradient centrifugation. The distribution of VSV 13 to 15 S RNA in the various cell fractions is shown in Table I.

RNA from each fraction was used to direct cell-free protein synthesis in extracts derived from wheat germ. Approximately equal amounts of VSV 13 to 15 S RNA, as measured by [3H]uridine counts, were added to each reaction mixture. The products of the reaction, labeled with [?S]methionine, were resolved on 10% polyacrylamide gels. Microdensitometer scans of the resulting radioautographs of the dried gels are shown in Fig. 4. Messenger RNAs isolated from free polyribosomes directed the synthesis of negligible or small amounts of glycoprotein, while the membrane RNA directed the synthesis of significant amounts of glycoprotein. Further, the RNA isolated from the postribosomal supernatant contained no mRNA capable of directing the synthesis of the VSV glycopro- tein.

800 600

Trypsm digest of [35S] methlonlne - labeled VSV N protein

cell -free product

I z r 250- I

VSV Marker

50

, i %A 0 0 0 IO 20 30 40 50 60 ?(

pH 3.5 cm

c Tryps~n digest of p5S] methionine-

000 labeled VSV NS protein

600 - cell-free product

VSV Marker

20 30 40 50 60 70 pm

pn 3.5 ._

/ Trypsln digest of [35S] methlonlne- I labeled VSV M protein

120 - I n I

cell - free

5 60 5

3, 40

t-

o 20

600 400b . I VSV marker 4

200

hl _ 0 0 0 IO 20 30 40 50 60 70

pH 3.5 cm

FIG. 3. Paper ionophoresis of [35S]methionyl tryptic peptides *T. G. Morrison, S. R. Weiss, L. E. Hightower, H. F. Lodish, M. A. derived from VSV proteins made in cell-free reactions. See Fig. 2

Bratt, in preparation. legend.

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from

6959

TABLE I

Distribution of VSV RNA in cell fractions

Each cellular fractionation is the result of a separate experiment. Column 1 shows the total counts in each fraction which is in WV 13 to

15 S RNA. Column 2 shows the percentage of the total counts of each fraction which is represented by VSV 13 to 15 S RNA. Column 3 represents the per cent of the total cellular VSV 13 to 15 S RNA present in each cell fraction.

Total $H counts in 13 to 15 S RNA

% of total o/o of

counts total 13

of each to 15s RNA in fraction

in 13 to various

15 s Cdl

RNA fractions

Continuous sucrose gradients High salt (buffer B)

Free polyribosomes High speed particulate

fraction (membranes) Postribosomal superna-

tant Buffer A

Free polyribosomes High speed particulate

fraction (membranes) Postribosomal superna-

tant Discontinuous sucrose gra-

dients Free polyribosomes Membrane Band 1 Membrane Band 2 Postribosomal supernatant

2.85 x lo5 30 40

1.74 x 105 26 24

2.60 x lo5

2.2 x 105 48

3.2 x lo5 25

2.5 x 105 33 32

7.8 x 10’ 39

1.45 x 105 58

6.5 x 10’ 52

1.5 x 105 44

30 36

28

40

18

33

15

34

By contrast, the mRNAs which encode the VSV N, NS, and M proteins were found in both free polyribosomes, in mem- brane-associated RNA, and in the supernatant fraction. In all experiments of this type, mRNA from the supernatant fraction directed the synthesis of appreciable amounts of M and NS proteins. The amount of nucleocapsid directed by supernatant RNA varied from preparation to preparation. In the experi- ment shown, RNA was used which directed very little nu- cleocapsid synthesis. Other preparations directed much larger amounts of nucleocapsid synthesis.

It may be argued that VSV glycoprotein messenger RNA was found exclusively in the membrane fraction of infected cells because free polyribosomes containing the glycoprotein mRNA are unusually large, and therefore, sedimented to the bottom of the gradient during centrifugation. This possibility seems unlikely since negligible A,,, material and no [3H]uridine- labeled material were found in the bottom 8 ml of the 3%ml gradient. Therefore, there is little possibility that large free polyribosomes pelleted in the sucrose gradient.

Alternatively, it may be argued that nonmembrane- associated polyribosomes containing the glycoprotein messen- ger RNA are found in the membrane fraction of infected cells because of nonspecific aggregation of the polysomes with membranes. Sucrose gradients containing high salt were used in an attempt to reduce such nonspecific aggregation. The RNA used in the experiment shown in Fig. 4 was isolated from polyribosome gradients containing high salt. (RNA isolated from gradients which contained low salt buffer directed the synthesis of the same products and in the same proportion as RNA isolated from gradients containing high salt.) There was

Membrane RNA

3

Free Polyrlbosomal RNA

No RNA

FIG. 4. VSV proteins made in cell-free reactions. Microdensitome- ter tracings of autoradiographs of polyacrylamide gels containing VSV proteins made in cell-free extracts of wheat germ. VSV RNA was prepared by Method A. Top, VSV proteins directed by VSV 13 to 15 S RNA isolated from the postribosomal supernatant. Middle, VSV proteins directed by VSV 13 to 15 S RNA isolated from the membrane fraction. Bottom, VSV proteins directed by VSV 13 to 15 S RNA isolated from free polyribosomes. Cell-free reactions (50 ~1) each contained 10 PCi of [%]methionine. Equal amounts of VSV RNA (as determined by cpm of [3H]uridine present in the RNA) were added to each reaction. This RNA was isolated from a high salt (Buffer B) sucrose gradient. The reactions were incubated at 22” for 120 min. The reactions were terminated by the addition of ribonuclease A. As calculated from a 5.~1 aliquot, the total incorporation of radioactivity into protein in the cell-free reaction (a) directed by supernatant RNA was 207,810 cpm; (b) directed by free polyribosomal RNA was 202,100 cpm; and (c) directed by RNA in the membrane fraction was 190,000 cpm. Reactions containing no added RNA incorporated 22,990 counts/ min into trichloroacetic acid-precipitated material.

some release of VSV 13 to 15 S RNA from the pellet of the polyribosome gradient when high salt was used (see Table I), but there was no specific mRNA released from the pellet when high salt buffer was used. Glycoprotein mRNA remains in the membrane fraction in high salt buffer.

Formamide-Polyacrylamide Gel Electrophoresis of VSV 13 to 15 S RNA Isolated from Three Cell Fractions-The above results concerning localization of the individual VSV mRNAs within the cell were obtained by translating mRNA isolated from various cell fractions in cell-free reactions. These results were confirmed by direct analysis of the VSV mRNA on formamide-polyacrylamide gels.

Rose and Knipe (14) have successfully resolved total VSV mRNA into four bands on formamide-polyacrylamide gels. Band 1 co-migrates with 28 S ribosomal RNA and is presum- ably the VSV 28 S mRNA. Knipe and Rose (32) have shown that Band 2 encodes the VSV glycoprotein, that Band 3 encodes the VSV N protein, and that Band 4 encodes the VSV NS and M proteins. Band 4 is composed of two separate species of RNA (14). These findings allow us to identify which RNA species are present in each cell fraction.

Aliquots of Wlabeled VSV 13 to 15 S RNA isolated from membranes, free polyribosomes, and the postribosomal super- natant fractions were subjected to electrophoresis on forma- mide-polyacrylamide gels. The gel was sliced horizontally, and the radioactivity present in each slice was determined by liquid scintillation counting as described under “Materials and Methods.” The results are shown in Fig. 5. All three fractions of VSV 13 to 15 S RNA lacked Band 1 RNA, which is expected

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from

6960

top

FIG. 5. Formamide-polyacrylamide gel analysis of VSV 13 to 15 S FIG. 6. Discontinuous sucrose gradients. The unfractionated cyto-

RNA isolated from three cellular fractions (Method A, Buffer A). VSV plasmic extract radioactively labeled with [3H]uridine (from 2.0 to 4.5

13 to 15 S RNA isolated from the supernatant, free polyribosomes, and hours postinfection), in the presence of actinomycin D (5 &g/ml) was

the membrane fraction of VSV-infected cells precipitated in 70% fractionated on a discontinuous sucrose gradient (Method B). -,

ethanol and resuspended in 200 ~1 of H,O. The supernatant RNA A

2o0 nm; O-O, [3H]uridine in trichloroacetic acid-precipitable

contained 2.5 x lo5 cpm; the free polyribosomal RNA, 2.2 x lo5 cpm; material (25-J aliquots of 1.2-ml fractions). The pellet contained 2.0

and the pellet RNA, 3.2 x lo5 cpm. Fifteen microliters of the x lo5 cpm; membrane Band 1 contained 2.5 x lo5 cpm; membrane

pellet-associated 13 to 15 S RNA (2.45 x 10’ cpm), 10 ~1 of the free Band 2, 1.25 x lo5 cpm; and the supernatant, 3.4 x 10Scpm.

polyribosomal RNA (1.18 x 10’ cpm), and 15 ~1 of the supernatant RNA (2.45 x 1O’cpm) were subjected to electrophoresis on formamide- while the material near the top of the gradient in the 30% polyacrylamide gels. The gels were sliced and each slice incubated in 3.5% NCS (Amersham/Searle) as described under “Materials and

sucrose layer (location of the cytoplasmic extract prior to

Methods.” The counting efficiency was 60%. Top, supernatant RNA. centrifugation) and at the interface between the 30% and 40%

Middle, RNA isolated from a membrane fraction. Bottom, free sucrose layer is considered to be the postribosomal superna- polyribosomal RNA. tant. In these gradients, there were two major membrane

bands. The bottom band at the interface between the 60%

since Band 1 RNA is probably the VSV 28 S species. RNA sucrose and the 45% sucrose layers contained the majority of

isolated from the membrane fraction contained Bands 2, 3, and the VSV RNA (membrane Band 1). There was a small amount

4. This result is consistent with earlier results which indicate of radioactivity associated with the membrane band at the

that this particulate fraction contains RNA which can direct interface of the 40% and 45% sucrose layers (Band 2). VSV 13 to

the synthesis of four VSV proteins. The RNA isolated from 15 S RNA was isolated from the four fractions. Of the

both free polyribosomes and the supernatant fraction con- [SHluridine-labeled material in the pellet, 39% was VSV

tained Band 3 and Band 4 RNA but is missing Band 2 RNA. 13 to 15 S RNA. Fifty-eight percent of the 3H-labeled material

Therefore, the failure of RNA isolated from free polyribosomes in membrane Band 1 was VSV 13 to 15 S RNA, and 52% of the

and the postribosomal supernatant to direct the synthesis of material in membrane Band 2 was VSV 13 to 15 S RNA. Forty-

the VSV G protein is due to the absence of the G mRNA in four percent of the supernatant material is VSV 13 to 15 S

these cell fractions. RNA. Most of the remainder of the RNA was in larger molec-

Fractionation of Cell Extracts on Discontinuous Sucrose ular weight material. There was a small amount of material in

Gradients-In the above experiments, we considered mem- the supernatant fraction that sediments between 7 and 4 S. The

brane-associated RNA as RNA found in the pellet of a velocity distribution of total cellular VSV 13 to 15 S RNA in the various

sucrose gradient run under conditions to display free polyribo- cell fractions is shown in Table I.

somes. Another method of preparing membrane fractions is by Aliquots of 13 to 15 S RNA isolated from each cell fraction

equilibrium centrifugation in discontinuous sucrose gradients, were analyzed on formamide-polyacrylamide slab gels (Fig. 7).

as described by Adelman et al. (37), Caliguiri and Tamm (38), The free polyribosome fraction contains RNA Bands 3 and 4

and Klenk et al. (27). In this procedure, the various membrane (encoding VSV N, M, and NS proteins), while negligible

fractions band in the sucrose layers according to their density. amounts of Band 2 RNA are found in this fraction. RNA

Rough endoplasmic reticulum is found at the 60% and the 45% isolated from membrane fractions contains Bands 3 and 4 as

sucrose interface and at the 45% and 40% sucrose interface; and well as Band 2, which encodes VSV G protein. RNA isolated

smooth endoplasmic reticulum and plasma membranes are from the postribosomal supernatant contains primarily Band 4

found in the 40% sucrose layer and in the 25% sucrose layer RNA. Thus, the distribution of VW 13 to 15 S mRNA in each

(27). Free polyribosomes are pelleted to the bottom of the tube. cell fraction is consistent with results obtained by the cell

A cytoplasmic extract of E&‘-infected cells labeled with fractionation on continuous sucrose gradients.

[3H]uridine was centrifuged to equilibrium in a discontinuous suaose gradient as described in “Method B” under “Materials DISCUSSION

and Methods.” Fig. 6 shows the A,,, pattern across the Cellular Localization of VSV 13 to 15 S RNA Species-There gradient as well as the profile of 3H-labeled VSV RNA. The is abundant evidence that proteins which are to be secreted pellet in this gradient contained the free polyribosomes (27), from the cells are made at least preferentially on membrane-

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from

IO 20 30 40 50

Gel Fraction

FIG. 7. Formamide-polyacrylamide gel analysis of VSV 13 to 15 S RNA isolated from discontinuous sucrose gradients (Fig. 6) was precipitated in 70% ethanol, and RNA from each cell fraction was resuspended in 0.2 ml of water. Free polyribosomal 13 to 15 S RNA contained 7.8 x lO’cpm, membrane Band 1 RNA contained 1.45 x lo5 cpm; membrane Band 2, 6.5 x 10” cpm; and supernatant RNA, 1.5 x lo5 cpm. Twenty microliters of each sample were subjected to electrophoresis on formamide-polyacrylamide gels as described under “Materials and Methods.” The gels were sliced, and each slice was treated with 3.5% Protosol as described under “Materials and Meth- ods.” The counting efficiency was 38%.

bound polyribosomes (3-6). However, generalization of this phenomenon to include membrane proteins may be an over- simplification. Lodish (9) and Lodish and Small (10) have found that two rabbit reticulocyte membrane proteins are made on free polyribosomes, and Lowe and Hallinan (11) have reported

the synthesis of rat liver NADPH-cytochrome c reductase, a membrane-bound enzyme, on free polyribosomes. We have used the VSV-infected mammalian cell as a model system for the study of the site of synthesis of membrane and nonmem- brane proteins.

Vesicular stomatitis virus proteins fall into two categories, those associated with cellular and viral membranes, and those not associated with membranes. Studies on the localization of VSV proteins within the cell have shown that VSV glycopro- tein and M proteins become associated with the membrane fraction of the cell following their synthesis (39, 40). The G protein comprises the spikes found on the outside of the lipid bilayer (19-21). M protein is found primarily on the inside of the lipid bilayer, although part of some molecules may penetrate to the outside of the bilayer (21). The VSV nu- cleocapsid protein and NS proteins are associated with the core of the virus (19-21) which is assembled in the cytoplasm of the infected cell, and only becomes associated with membrane as the virus buds from the surface of the cellular membrane which has been modified with viral G and M proteins. Thus, the VSV

6961

N and NS proteins may be considered model cytoplasmic proteins or nonmembrane proteins.

In order to determine the cellular location of each VSV mRNA, we isolated VSV 13 to 15 S RNA from three cellular fractions: the membrane fraction, the nonmembrane or free polyribosomal fraction, and the postribosomal supernatant. We have used two different fractionation procedures, and have obtained similar results with both procedures. A postnuclear cell supernatant was fractionated by velocity linear sucrose gradients containing either low (0.025 M) or high (0.5 M) salt. Under these conditions, the membrane fraction is found in the pellet, and the free polyribosomes are found in the middle of the gradient. We have also centrifuged this cell extract to equilibrium in a discontinuous sucrose gradient. In this gradi- ent, the free polyribosomes are pelleted, while the membrane fractions band at their appropriate density (Fig. 6).

In both procedures, the fraction of labeled VSV 13 to 15 S RNA recovered in the postribosomal supernatant was very similar (between 32% and 36% of the total VSV 13 to 15 S RNA; Table I). The amount of VSV 13 to 15 S RNA recovered from free polyribosomes purified by the equilibrium gradient tech- nique was somewhat less than that from the free polyribosomes purified by the velocity sucrose gradients (18% compared to 28%). Cioli and Lennox suggested that when velocity gradients are run so as to pellet membranes, polyribosomes with very small amounts of membrane are not pelleted (7). Thus, free polyribosomes from the velocity gradients might be con- taminated with membrane-associated polyribosomes. Cioli and Lennox have suggested that discontinuous sucrose gradients yield better estimates of the amount of membrane-bound

material. This explanation may account for the fact that there is more VSV 13 to 15 S mRNA in the free polyribosomes re- covered from the velocity gradients.

The 13 to 15 S RNA isolated from each subcellular fraction was translated in a cell-free system. In addition, we directly identified the individually labeled mRNA species present by formamide-polyacrylamide electrophoresis. We found that the messenger RNA for the glycoprotein is located almost exclu- sively in the membrane fraction of the cell. These results suggest that the G protein is synthesized on membrane-bound polyribosomes. Presumably, the nascent G protein is trans- ported through the rough endoplasmic reticulum in a manner similar to that of proteins which are to be excreted from the cell. If the G protein is localized on the inner surface of the endoplasmic reticulum membrane, then fusion of the endo- plasmic reticulum vesicles with the plasma membrane would result in the G protein being on the outside of the plasma membrane bilayer.

The messenger RNA which encodes the other viral mem- brane protein-the M protein-is localized both in the mem- brane fraction and in the free polyribosomes. The main evi- dence for this comes from the cell-free protein synthesis ex- periments (Fig. 4); the mRNAs for M and NS protein co- migrate on the formamide gels used (Fig. 5), and have not been resolved on any gel system. We also found that the mRNAs which encode the VSV nonmembrane proteins-N and NS- are also located in both the membrane and free polyribo- somal fractions. It is possible that all of the M, N, and NS proteins are made on free polyribosomes, and some of these polyribosomes attach artifactually during rupture of the cell. Alternatively, these proteins may be synthesized on both free and membrane-bound polyribosomes.

The absolute specificity in the location of any mRNA may

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from

6962

occur only for mRNAs which encode proteins such as G, which must be transported across cell membranes. The VSV M protein is found on the inner side of the cell membrane and, therefore, is probably not transported across cell membranes. In this connection, it is relevant to point out that two reticulocyte membrane proteins which are found-like M protein-on the inner surface of the plasma membrane are also made on free polyribosomes (11).

Nontranslated Messenger RNA-We have found that not all

the VSV 13 to 15 S RNA present in the cell is being translated. Between 32% and 36% of the total 13 to 15 S RNA is in the

postribosomal supernatant. This RNA is fully capable of being

translated. We have isolated this RNA from the infected cell and used it to direct the synthesis of authentic VSV proteins in a cell-free system.

The most likely explanation for the failure of the cell to translate this RNA is that VSV messenger RNA is produced in such quantities that the protein synthetic machinery of the

infected cell becomes saturated. The excess messenger RNA is thus found in the postribosomal supernatant.

In most preparations of supernatant RNA, the predominant species of RNA present is the M protein and the NS protein messenger RNA. Glycoprotein messenger RNA is never found in this fraction. The unequal distribution of the different VSV 13 to 15 S RNA species in this fraction may reflect the relative

concentration of each messenger in the cell and/or the different affinity of each messenger for the host cell ribosomes.

1. Siekowitz, P., and Palade, G. (1960) J. Biophys. Biochem. Cytol. 7, 619-625

2. Rosbash, M., and Penman, S. (1971) J. Mol. Biol. 59,227-241 3. Hicks, S. J., Drysdale, J. W., and Munro, H. N. (1969) Science 164,

584-585 4. Redman, C. M. (1969) J. Biol. Chem. 244,4308-4315 5. Ganoza, M. C., and Williams, C. A. (1969) Proc. N&l. Acad. Sci. U.

S. A. 63,1370-1376 6. Campbell, P. N. (1970) FEBS Lett. 7,1-7 7. Cioli, D., and Lennox, E. S. (1973) Biochemistry 12,3211-3217 8. Whaley, W. G., Dauwalder, M., and Kephart, J. E. (1972) Science

REFERENCES

9. 10. 11. 12.

13

14. 15.

16.

17. 18.

19.

20. 21.

22. Emerson, S. U., and Wagner, R. R. (1973) J. Virol. 12,1325-1335 23. Mudd, J. A., and Summers, D. F. (1970) Virology 42,328-340 24. Kingsbury, D. W. (1973) J. Viral. 12,1020-1027 25. Grubman. M. J.. and Summers. D. F. (1973) J. Viral. 12.265-274 26. Milcarek,‘C., and Penman, S. (1974) J: Mel: Biol. 89,32?-338 27. Klenk, H.-D., Wijllert, W., Rott, R., and Scholtissek, C. (1974)

28. Roberts, B. E., and Paterson, B. M. (1973) Proc. N&l. Acad. Sci.

29. 30. 31. 32. 33. 34. 35. 36.

37.

38. 39.

40. David, A. E. (1973) J. Mol. Biol. 76, 135-148

175,596-599 Lodish, H. F. (1973) Proc. N&l. Acad. Sci. U. 5’. A. 70,1526-1530 Lodish, H., and Small, B. (1975) J. Cell Biol., in press Lowe, D., and Hallinan, T. (1973) Biochem. J. 136, 825-828 Huang, A. S., Baltimore, D., and Bratt, M. A. (1971) J. Viral. 7,

389-394 Huang, A. S., Baltimore, D., and Stampfer, M. (1970) Virology 42,

946-957 Rose, J., and Knipe, D. (1975) J. Viral. 15,994-1003 Follett, E. A. C., Pringle, C. R., Wunner, W. H., and Skehel, J. J.

(1974) J. Virol. 13,394-399 Morrison, T., Stampfer, M., Baltimore, D., and Lodish, H. F.

(1974) J. Viral. 13,62-72 McShariv. J. J.. and Wagner. R. R. (1971) J. Viral. 7.412-415 McSharry; J. J., Comp&, ‘R. W.,‘and’Choppin, P. W. (1971) J.

Viral. 8,722-729 Cartwright, B., Talbot, P., and Brown, F. (1970) J. Gen. Virol. 7,

267-272 Howatson, A. F., and Whitmore, G. F. (1962) Virology 16,466-478 Moore, N. F., Kelley, J. M., and Wagner, R. R. (1974) Virology 61,

292-296

Virology 57,28-41

U. S. A. 70,2330-2334 McDowell, M. J., and Joklik, W. K. (1971) Virology 45,724-733 Lodish, H. F., and Desalu, 0. (1973) J. Biol. Chem. 248, 3520-3527 Duesberg, P. H., and Vogt, P. K. (1973) J. Viral. 12,594-599 Knipe, D., Rose, J., and Lodish, H. F. J. Viral. 15,1004-1011 Lodish, H. F. (1968) Nature 220,345-350 Goldman, E., and Lodish, H. F. (1971) J. Viral. 8,417-429 Bretscher, M. S. (1971) Nature New Biol. 231,229-231 Roberts, B. E., Mathews, M. B., and Bruton, C. J. (1973) J. Mol.

Biol. 80,733-742 Adelman, M. R., Blobel, G., and Sabatini, D. D. (1973) J. Cell Biol.

56,191-205 Caliguiri, L. A., and Tamm, I. (1970) Virology 42,100-111 Wagner, R. R., Snyder, R. M., and Yamazaki, S. (1970) J. Viral. 5,

548-558

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from

T G Morrisonvirus.

Site of synthesis of membrane and nonmembrane proteins of vesicular stomatitis

1975, 250:6955-6962.J. Biol. Chem. 

  http://www.jbc.org/content/250/17/6955Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/250/17/6955.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on Decem

ber 26, 2018http://w

ww

.jbc.org/D

ownloaded from