Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and El, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and El are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion. KEY WORDS membrane glycoprotein insertion Sindbis virusThe biosynthesis of integral membrane glycoproteins has been studied using enveloped viruses such as VSV and Sindbis virus as model systems (10,15,24,21). The advantage of using such systems is that many viruses have few structural proteins, usually only one or two integral membrane proteins and, in some cases, an internal peripheral protein. In contrast, an average mammalian cell contains several hundred membrane proteins. Further, these viruses do not contain enough genetic information to encode all of the enzymes necessary for their biogenesis and therefore must rely on the host cell for many functions.In general, host cell protein synthesis is inhibited during viral infection, making the study of specific proteins much easier.Studies with VSV and Sindbis virus have dem- 154J. CELL BIOLOGY 9 The Rockefeller University Press 9
Studies in this laboratory on the lipopolysaccharide surface layer of the enteric bacteria have centered around' the Salmonella anatum 0-antigen. The purpose of these studies has been to define the mechanisms by which bacterial and bacteriophage genomes interact to bring about the formation of the specific polysaccharide antigen of the cell surface. Our previous work'-3 defined structural changes produced in the S. anatum lipopolysaccharide by bacteriophages E15 and eY4. These changes consist primarily, following infection by e15, in the synthesis of a A rather than an a-D-galactosyl linkage in the galactosyl-mannosyl-rhamnose repeating unit of the antigen, and following infection by C34, in the addition of glucose to the antigen. These studies also showed that the synthesis of the bacterial enzyme 0-10 transacetylase is repressed by e15 and that the mechanisms for the synthesis of UDPG and TDPRh are not observably modified by the phages.4' 5 Since it seemed likely that the el' phage genome was involved primarily in controlling or modifying the complex transglycosylating enzyme or enzymes that bring about the synthesis of the repeating galactosyl-mannosyl-rhamnosyl sequence of the polysaccharide, it was necessary to develop a system to study this complex enzymatic reaction. Such a system, described in the present paper, is rather different from the lipopolysaccharide transglycosylases described in work from other laboratories,6-8 and the implications suggested by the differences are discussed.Materials and Methods.-The bacterial strains and procedures used for isolating and analyzing lipopolysaccharide have been described in previous papers.'-3The sources of nucleotide sugars were as follows. GDPM and UDPG were purchased from Sigma Chemical Co. and Calbiochem, respectively. TDPRh was prepared by the method of Bernstein and Robbins.5 UDPG-C14 was made by the procedure of Wright and Robbins9 from glucose-fructose-C'4 with an enzyme fraction from bakers' yeast. UDPGal and UDPGal-C14 were prepared by the method of Wiesmeyer and Jordan'0 with the modification that the concentrated 70% ethanol extract was purified by hanging strip paper electrophoresis with a triethylamine-acetic acid buffer.9 UDPG-Gal-C'4 was the equilibrium mixture of UDPG and UDPGal prepared from UDPG-C14 by treatment with an epimerase preparation from galactose-grown E. coli K12. UDPGal-G-C'4 was an equilibrium mixture prepared in the same way from UDPGal-C'4. In both cases equilibration was checked by mild acid hydrolysis followed by paper chromatography in n-butanol-pyridine-water (6:4:3). This chromatographic system separates glucose and galactose clearly.Incorporation of C'4 from a nucleotide sugar into cell wall-membrane fractions was measured by treating 0.1-0.2-ml reaction mixtures with 0.2 ml of 2 N acetic acid to stop further reaction and filtering the material through 0.45-M type B-6 membrane filters (S.-and-S.). The filters were washed repeatedly with, 1 N acetic acid and water and attached to aluminum planchets for counting. The absolut...
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