The effects of tunicamycin on the synthesis and export of fat body proteins and glycoproteins in larvae of the greater wax moth Galleria mellonella (L.)

Insect Biochem., Vol. 12, No. 3, pp. 301 309. 1982 Printed in Great Britain

0020-1790/82/030301-09503.00/0 © 1982 Per qamon Press Ltd.

THE EFFECTS OF T U N I C A M Y C I N O N THE SYNTHESIS A N D E X P O R T OF FAT B O D Y P R O T E I N S A N D G L Y C O P R O T E I N S IN LARVAE OF THE GREATER WAX M O T H G A L L E R I A M E L L O N E L L A (L.)* STEPHEN G. MILLER'I"and DONALD L. SILHACEK Insect Attractants, Behavior, and Basic Biology Research Laboratory, Agricultural Research, Science and Education Administration, USDA, Gainesville, FL 32604, U.S.A. (Received 23 March 1981: revised 1 September 1981

Abstract--Treatment of fat body from the greater wax moth, Galleria mellonella (L.), with tunicamycin in vitro caused complex alterations in protein synthesis. These included a 5000 inhibition of protein synthesis and a greater than 90°,,o inhibition of both protein release and glycoprotein biosynthesis. The inhibition of glycoprotein biosynthesis can be accounted for by the well-documented effects of tunicamycin on GlcNAc-pyrophosphoryl dolichol synthesis, while the two additional effects are likely to have a complex basis, Synthesis and export of the four storage proteins was also studied in fat body treated with tunicamycin. Exposure to antibotic for short periods of time (2.5 hr) resulted in the synthesis of lower mol. wt variants of at least two of these polypeptides in treated cultures, both of which normally contain carbohydrate. Treated fat body released only putatively fully-glycosylated forms of the storage proteins which suggests that the attachment of carbohydrate is in some way necessary for their secretion. Newly synthesized storage proteins were not detected in fat body treated with tunicamycin fo- longer time periods (approx. 7 hr), which indicates carbohydrate may also increase the intracellular half life of this class of proteins. Key ~brd Index: Galleria mellonella, fat body. protein biosynthesis, tunicamycin, storage proteins

INTRODUCTION ONE OF THE most conspicuous functions of larval fat body in holometabolous insects is the biosynthesis, transport, and sequestration of the so-called storage proteins (see THOMSON, 1975 and WYATT and PAN, 1978 for reviews). These proteins accumulate in large quantity in larval haemolymph, and ultimately come to reside in pharate-pupal and pupal fat body where they are stored as proteinaceous spheres or granules (ToJo et al., 1978; 1980). Virtually nothing is known of the manner in which fat body regulates this bidirectional transport of storage proteins in a developmental stage-specific sequence. Studies in our laboratory have been aimed at elucidating basic features of storage protein synthesis, metabolism and transport in the greater wax moth, Galleria mellonella (L.). Four storage polypeptides have been identified in both haemolymph and fat body of this moth during the final larval instar as well as in pupae (MILLER and SILHACEK, 1982a). Ontogenetically-distinct dual roles served by fat body in the export and sequestration of these storage proteins have been established, specifically: (1) larval, but not

* Mention of a commercial or proprietary product does not constitute an endorsement by the USDA. 1"Postdoctoral Fellow employed through a cooperative agreement between the Department of Entomology and Nematology, University of Florida and the Insect Attractants, Behavior, and Basic Biology Research Laboratory, Agricultural Research, Science and Education Administralion, USDA, Gaint:sville, FL 32604, U.S.A.

pupal, fat body cultured in citro both synthesizes and secretes all four polypeptides: and (2) pharate-pupal and pupal fat body sequesters the storage proteins from haemolymph in vivo (MILLER and SILHACEK, 1982b). These findings indicate that tt'e storage proteins possess structural features distinct from other cellular proteins which are recognized by fat body at discrete stages of development. Three of the four storage proteins in G. mellonella have 9een shown by indirect analyses to contain both mannose and Nacetylglucosamine (GlcNAc), carbohydrates typicall2~ associated with glycoproteins destined for secretion (KORNFELD and KORNFELD, 1976), The function(s) served by carbohydrate in the intracellular transport (OLDEN et al., 1978: OLDEN et al., 1979), secretion (STRUCK et al., 1~78: LOH and GAINER, 1978: DUKSIN and BORNSTEIN, 1977: SCHWAIGER and TANNER, 1979), and function (FUJISAWA et al., 1978; SCHWAtGER and TANNER, 1979) of specific glycoproteins in a number of plant and animal species has been inferred, These studies have been facilitated by the exposure of cell and tissue cultures to the naturally-occurring antibiotic, tunicamycin (TAKATSUKI and TAMURA, 1971). Tunicamycin indirectly inhibits the glycosylation of proteins by interfering with the transfer of GlcNAc phosphate from U D P - G I c N A c to dolichol phosphate TAKATSUKI and TAMURA, 1971: TAKATSUKI et al., 1975: TKACZ and LAMPEN, 1975; STRUCK and LENNARZ, 1977). Currently available preparations of this antibiotic also contain at least one additional major component which inhibits protein synthesis by a mechanism not yet identifed (MAHONEY and DUKSIN, 1979).

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The present study examines some of the effects of tunicamycin on the synthesis and transport of fat body proteins in G. mellonella. There were two objectives. First, to determine whether tunicamycin exerts any effect on total protein and glycoprotein synthesis in light of the fact that to our knowledge, exposure of insect tissues to this drug has not yet been reported. And second, to ascertain whether treated fat body continues to synthesize and secrete the four storage proteins. Tunicamycin exerted several complex effects on fat body treated in vitro including a rapid inhibition of radiolabelled leucine and GIcNAc incorporation into b o t h released and retained proteins. Fluorographic analyses of immunoprecipitated storage proteins revealed that antibiotic treatment induced the synthesis of lower mol. wt variants. These putatively non-glycosylated storage proteins were not released which suggests that c a r b o h y d r a t e confers at least one c o m p o n e n t of the specificity required for their mobilization and transport from fat body.

ditions [~'H]- and [14C]-labelled samples were coumcd with efficiencies of 43 and 89!!,,. respectively. Specific proteins synthesized by cultured fat bod) were assessed by fluorographic analyses of labelled polypeptides separated on sodium dodecyl sulphate (SDS) polyacrylamide gels. In these studies fat body was incubated in the presence of [35S]-methionine (lll0Ci/mmol: New England Nuclear) and retained and released protein fractions recovered by immunoprecipitation (below~.

Antisera and immunoprecipitation oJjdt body proteins

Final instar larvae at day 6 of development (MILLER and SILnACEK, 1982a) were used in these studies. The paired perivisceral fat bodies from surface-sterilized larvae were dissected into chemically defined Grace's medium (Gibco) that contained 50 units penicillin-streptomycin/ml. Fat body was cultured at 27°C in plastic Petri dishes which contained 1 ml medium.

The preparation of antiserum to the 82 K M r storage protein which reacted also with the 81 K protein has been described (MILLER and SILHACEK, 1982a). A mixture of antibodies to the 74 and 76 K Mr storage proteins were raised in the same fashion and their specificity verified using both the Ouchterlony double-diffusion test and SDS polyacrylamide gel analyses of precipitated fat body and haemolymph proteins (MILLER, unpublished). Antisera and unlabelled carrier storage protein (0.01 mg/ ml) were added either to incubation media or to fat body homogenates containing radiolabelled storage proteins prepared as described above. These mixtures were incubated at 4°C for 16 hr and the immunoprecipitates were collected by centrifugation at 15000 (20rain at 4°C) through homogenization buffer that contained 1.0M sucrose, 1°,o (w/v) Triton X-100 (Packard) and l'!,, (w/v) sodium deoxycholate (Sigma). The pellets were washed twice with 0.05 M potassium phosphate, pH 7.4 that contained 0.5 M NaCl, l mM EDTA, and I mM phenylmethyl sulphonyl fluoride. These samples were dissolved in 0.2 M Tris-HCl, pH 6.8 containing l°~; (w/v) dithiothreitot, 4",, (w/v) SDS and 40%0 (w/v) glycerol and then diluted with three volumes of homogenization buffer.

Radioactice biosynthetic labellin9 o f f at body proteins in

SDS polyacrylamide (4el electrophoresis and fluoroqraphy

vitro Previously cultured or freshly dissected tissues were rinsed in two changes of sterile Grace's medium lacking the amino acid used for incorporation prior to labelling. Paired fat body lobes were then cultured in the presence of radioisotope at 27°C with constant agitation. Tunicamycin (generously provided by Dr. Robert Hamill, Eli Lily and Co.) was added to culture media prior to the addition of tissues. Stock solutions of antibiotic were prepared by dissolving the crystalline material in a minimal volume of 1 M NH4OH and then adjusting the concentration to 1 mg/ml with deionized water. Solutions were filter-sterilized and stored in vials at - 2 0 ° C prior to use. The incorporation of L-[4,5-H3]-leucine (5 Ci/mmol: New England Nuclear)and N-[acetyl-l-~'*C]-GlcNAc (50 mCi/mmol; New England Nuclear) into fat body proteins was determined to provide estimates of total protein and glycoprotein biosynthesis, respectively. Radiolabelled protein fractions were recovered from both cultured fat body (retained fraction) and the medium in which they had been incubated (released fraction) and prepared for liquid scintillation counting. Soluble retained proteins were obtained from the 1000g supernatant (10min at 4:C) of tissues homogenized in an equal volume of homogenization buffer which was 0.05 M Tris-HCl, pH 8.0 containing 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethyl sulphonyl fluoride and 0.01°4 l-phenyl-2-thiourea. Released proteins were obtained from media to which 0.1 mg ovalbumin had been added as carrier for precipitation. Acid-insoluble radiolabelled proteins were prepared from both fractions by precipitation with an equal volume of 105o trichloroacetic acid (TCA) in an ice bath for 120 min. Precipitates were collected onto GF-B glass fibre filters (Whatman, Ltd.) with suction and washed three times with 5 ~ (w/v) TCA. The filters were dried at 150°C, added to 10 Aquascint (I.C.N.) and the radioactivity determined using a Packard ® scintillation spectrometer. Under these con-

Protein samples were prepared for electrophoresis by heating at 100~'C for 3 min in a solution which contained 0.5~o (w/v) dithiothreitol, 1~0 (w/v) SDS and Y,'o (w/v) glycerol. These reduced and denatured proteins were separated on 7.5~.0 polyacrylamide slab gels (SWANTON et al.. 1975) containing 0.1~o SDS. Gels were simultaneously fixed and stained in 50}o (w/v) TCA containing 0.5~% Coomassie brilliant blue ® R-250 (BioRad), then destained in methanol:acetic acid:water (5:1:5, by vol) overnight. Radiolabelled proteins were detected fluorographically by impregnation of the gel in ENHANCE (New England Nuclear) followed by several rinses in deionized water. The gels were dried in vacuo at 70~C and exposed to prefogged (LASKEV and MILLS, 1975) Royal O-mat XR-5 X-ray film IKodak) at -70':C.

MATERIALS AND METHODS

Animals and culture of fat body

RESULTS

The effects of tunicamycin on the synthesis of total fat body proteins and 91ycoproteins The incorporation of [3H]-leucine into TCA-precipitable fat body proteins was used to assess the effects of tunicamycin on protein synthesis. Although a m a j o r c o n t a m i n a t i n g c o m p o n e n t of tunicamycin mediates this effect (MAnONEV and DUKSiN, 1979), the fact that non-glycosylated cellular proteins turn over more rapidly than those containing their normal complement of c a r b o h y d r a t e (HAsILIK a n d TANNER, 1978: SCHWAIGER and TANNER, 1979) may also be a contributing factor. In preliminary experiments, paired fat body lobes were exposed to a range of tunicamycin concentration~ (1-20/ag/ml) for 2 4 h r and then labelled for an additional 5 hr in the presence of antibiotic (Fig. 1). Incorporation of [3H]-leucine was

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Fig. 1. The effect of tunicamycin concentration on the incorporation of [3H]-Ieucine into total fat body proteins. Pairs of tissues were cultured for 24hr in either 1 ml Grace's medium or 1 ml medium containing the indicated amounts of tunicamycin. Treated and control fat bodies were then labelled for 5 hr in 1 ml Grace's medium (lacking leucine) with 2 ~Ci [3H]-leucine. and total TCA-precipitable radioactivity were determined in the retained (eO) and released ( O - - © ) protein fractions. Each point represents the average of two determinations and bars correspond to ranges.

actually stimulated by 15-307o at concentrations of tunicamycin below 10~g/ml, but was inhibited 50% relative to control tissues at antibiotic concentrations of 10-20/~g/ml. Higher antibiotic concentrations did not reduce incorporation into retained proteins below this level (data not shown). The time-course of the inhibition of [3H]-leucine incorporation was determined using tissues exposed to tunicamycin for periods of 5-24.5 hr (Table 1). Incorporation into proteins retained by the fat body

was inhibited approx. 30°,o by 9 hr and 35",, by 17.5 hr of exposure. Control and treated fat body which had been cultured for up to 16 hr also exhibited different initial rates of incorporation over the first hr of labelling (Fig. 2a). In agreement with the time-course studies (Table 1), the amount of TC.&-precipitable label accumulated in treated tissues by 6 hr of labelling was approx. 50°;, of that recovered in controls (Fig. 2a). The incorporation of [m'~C]-GIcNAc into total retained fat body proteins was used to assess the effects of tunicamycin on glycoprotein biosynthesis. Tissues pre-incubated in the presence of antibiotic exhibited a greater than 90'!o reduction in incorporation relative to untreated fat body C?able 1). This effect of tunicamycin was both more pr,3nounced and more rapid than the inhibition of t o t a protein synthesis, and was apparent within 5 hr of exposure. Control tissues labelled with [14C]-GIcNAc subsequent to 15 hr of culture incorporated label into retained proteins at a high rate which was preceded by a 2 hr lag (Fig. 2ct. In contrast, tissues exposed to 20/~g/ml tunicamycin for 10 hr and then labelled for up to an additional 6 hr, incorporated this radiolabelled precursor into protein at a much lower rate which was preceded by a 3 hr lag. Although the primary effect of tunic~mycin in inhibiting both protein synthesis and the attachment of GlcNAc phosphate to dolicholphosphate has been established in cell-free systems, it has al',~o been shown to exert secondary effects on membrane transport which manifests itself in the inhibition of metabolite uptake (OLDEN et al., 1979). Therefore, it was important to determine whether the apparent inhibition of glycoprotein synthesis in treated fat body could be explained as a primary effect of tunicamycin or whether it was a secondary effect on GIcNAc uptake. To test this, control tissues and those cultured in the presence of 20/lg/ml tunicamycin for 15 hr were incubated with [14C]-GIcNAc for up to an additional 4 hr, then the total radioactivity recovered in whole tissues was determined (Table 2). The uptake of label by both tissue types was approx, linear for at least 4hr, and in addition, the absolute r~tdioactivity/mg tissue recovered at any given time point were indis-

Table 1. Time-course of tunicamycin effects on the incorporation of [3H]-leucine and [t4C]-GIcNAc into fat body proteins* Time+ (hr) 5 9 17.5 24.5

dpm/mg Tissue (",, control) Retained Released [3H]-leucine [14C]-GIcNAc [3H]-leucine [14C]-GIcNAc 92 (61 71(11) 64(4) 72(8)

7 (4) 8(2) 6(3) 13(2)

33 (6) 8(2} 10(3) 12(3)

11 (31 5(21 9(11 20(9)

* Fat bodies were cultured in 1 ml Grace's medium containing either distilled water or 20,ug/ml tunicamycin. At various times treated tissues were transferred to fresh media containing tunicamycin, 1.0#Ci/ml [3H]-leucine and 0.5~Ci/ml [~4C]-N-acetylglucosamine and incubated for 5 hr. TCA-precipitable radioactivity per mg was determined for retained and released protein fractions and is expressed here as ",, control values at each time point. Numbers in parentheses refer to ±S.E.M. of at least three separate determinations. + Times correspond to the total duration of exposure to tunicamycin including the 5 hr labelling interval.

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Fig. 2. Time-course of [3H]-leucine and [~4C]-GIcNAc incorporation into total fat body proteins from control tissues ( ' - - ' ) and those exposed to tunicamycin (O---O). Paired tissues were cultured for 10 hr either in 1 ml Grace's medium or medium containing 20/Jg/ml tunicamycin. The tissues were then cultured in 1 ml Grace's medium (lacking leucine) containing either 1 #Ci [aH]-Ieucine/ml or 0.5/~Ci [~4C]-GIcNAc/ml for the indicated times. Incorporation of [aH]-Ieucine into total TCA-precipitable retained and released fat body proteins are shown in (a) and (b), respectively. Incorporation of [14C]-GIcNAc into total TCA-precipitable retained and released fat body proteins are shown in (c) and (d), respectively. Each point represents the mean of at least three determinations and bars correspond to + S.E.M. tinguishable. These results strongly suggest that in this system, the greater than 90% reduction in GIcNAc incorporation mediated by tunicamycin was due to the inhibition of protein glycosylation at the level of GlcNAc-pyrophosphoryl-dolichol synthesis.

The effects of tunicamycin on the export of fat body proteins Larval fat body cultured in vitro secretes approx. 20 polypeptides of which the four storage proteins are by far the most abundant (MmLER and SmXACE~¢, 1982b). The export of total TCA-precipitable [3H]-leueinelabelled proteins was inhibited in a dose-dependent fashion by tunicamycin (Fig. 1). Following a 24hr exposure t o ~ i b i o t i c , export by tissues at 20/~g/ml tunicamyein .Mrs inhibited greater than 80%. Experiments in which fat body was exposed to 20/~g/ml tunicamycin for 5-24.5 hr and labelled with either [3H]-ieucine or [14C]-GIcNAc revealed that the incorporation of both precursors into the released pro-

tein fraction was inhibited well before 24 hr (Table 1). Both [3H]- and [14C]-radioactivity per mg tissue recovered in the media were reduced by more than 90% relative to controls within 9 hr of treatment. As was found to be the case with proteins in the retained protein fraction (Table 1), the incorporation of GIcNAc was inhibited to a lesser extent in tissues exposed to tunicamycin for up to 24.5 hr. This effect may be due to metabolism of antibiotic by cultured fat body since GIcNAc incorporation by freshly dissected tissue which had been transferred to tunicamycin-eontaining medium in which fat body had been cultured for 19 hr was inhibited by only 10% (data not shown), The inhibition of release of proteins labelled with both radiolabelled precursors is clearly seen in experiments in which fat body was cultured for 10 hr (with or without 20/~g/ml tunicamycin) and then labelled for up to an additional 6 hr (Fig. 2). Incorporation of [3H]-leucine into exported protein was roughly linear for at least 6hr in both control and

305

Fat body protein biosynthesis Table 2. Uptake of [~'*C]-GIcNAc by control tissues and those exposed to tunicamycin Time Ihrl 1 2 4

Total ~'*C-dpm* Control Tunicamycin 398 (+83) 803 ( _+53) 1426 (+97)

470 (_+92) 867 ( -+89) 1609 ( -+ 106)

* Fat bodies were cultured for 15hr in 1 ml Grace's medmm containing either distilled water or 20 lag/ml tunicamyciu. 0.5 ~Ci/ml [~4C]-GIcNAc was then added and uptake proceeded for the indicated lengths of time: labelled tissues were rinsed twice in unlabelled medium and then added to 1 ml tissue solubilizer: total dpm for each sample was determined as described in Materials and Methods. The + S.E.M. of three separate determinations are given in parentheses. treated tissues (Fig. 2b), but recovery of level from the control media was much greater. Similarly, control tissues labelled with [14C]-GIcNAc released TCAprecipitable ~C-activity into the medium at a high rate which was preceded by a 3 hr lag (Fig. 2dL In contrast, the recovery of ~4C-activity from media in which treated tissues had been cultured was negligible during the course of at least 6 hr of labelling (Fig. 2d). The ~q:fects of tunicamycin on the synthesis o f the stora qe proteins G. mellonella larval fat body from the stage of development used in this study synthesizes and exports four storage proteins when cultured in vitro (MILLER and SILHaCEK, 1982bL The storage polypeptides having tool. wts of 81 and 82 K daltons on SDS polyacrylamide gels are antigenically and structurally similar but only the latter protein contains detectable carbohydrate (MILLER and SILHACEK,1982aL The 74 and 76 K M r storage polypeptides both contain carbohydrate but are similar neither to each other nor to the 81/82 K M r polypeptides by these same criteria. These observations suggested that exposure of fat body to levels of tunicamycin sufficient to inhibit GlcNAc incorporation would result in the synthesis of lower mol. wt variants of all except the 81 K M~ storage protein. Fluorography of [3sS]-methioninelabelled fat body proteins immunoprecipitated with antisera specific for the 81/82 K Mr and 74/76 K (Fig. 3t rvl~ proteins showed this to be the case. A diffuse band corresponding to the 82 K M~ polypeptide was seen in immunoprecipitates of control fat body {data not shown) in spite of the poor resolution typically obtained between it and the 81 K M~ polypeptide (MILLER and SILHACEK, 1982a). This same band was missing in immunoprecipitates of tissues exposed to 20/~g/ml tunicamycin for as short a time as 2.5 hr. Times of exposure to antibiotic of 5 hr (lane 41 and 7.5 hr (lane 5) resulted in correspondingly reduced levels of the 81 K Mr polypeptide (data not shown). Clear-cut results were obtained from immunoprecipitation of control and treated fat body proteins with antiserum specific for the 74 and 76 K M, storage proteins (Fig. 3). These fluorographs show that control fat body synthesizes two immunoreactive polypeptides dane 2) whose mobilities coincide with the positions of purified and radiolabelled 74 and 76 K stan-

dards (lane 1). The proteins immunoprecipitated from homogenates of fat body exposed to 20 #g/ml tunicamycin for 2.5 hr included not only the 74 and 76 K Mr proteins but also two polypeptides hazing apparent mol. wts of 71 and 72 K (lane 3). After a 5 hr exposure to antibiotic only the 71 and 72 K Mr polypeptides were recovered in immunoprecipitates (lane 4). Radiolabelled proteins were not recovered from immunoprecipitates of tissues treated with turicamycin for times longer than 5-7 hr (data not shown). The lower mol. wt variants o f the 74 and "76 K Mr polypeptides synthesized by treated tissues are not released

The proteins released into medium in which control and tunicamycin-treated fat body had been cultured were also immunoprecipitated with anttsera. The recovery of radiolabelled protein from treated cultures was both low and of limited duration (Fig. 4). This was particularly true of the 81 and 82 K polypeptides whose export virtually ceased within 2.5 hr of e x posure (lanes 1-3). Immunoprecipitation of media in which treated fat body had been cultu'ed using the 74/76 K antiserum, however, revealed ~:hat only the normal mol. wt forms of these proteins were released (lane 5). No detectable polypeptides were recovered from media in which fat body had been cultured for 5 hr in the presence of antibiotic (lane 6). Significantly, the two lower mol. wt variants present in the retained protein fractions (Fig. 3b, lanes 3 and 4) were not released. DISCUSSION The most straightforward effect of tunicamycin on cultured larval fat body from G. mellonella in the present study was its inhibition of radiolabelled GIcNAc incorporation into TCA-precipitable proteins. Exposure of paired lobes of tissues to 20/~g/ml of this antibiotic resulted in the virtually complete (less than 10°; of control tissues) and rapid (within 5 hr) cessation of the synthesis of GlcNAc-containing glycoproteins. Tunicamycin has been reported to reduce the incorporation of radiolabelled sugars into specific glycoproteins in non-insect systems as well (HICKMAN et al., 1977: GAHMBERG et al., 1980: OLDEN et al., 1979; MILLER et al., 1980; STRICKLER and PATTON, 1980: HOUSLEY et al., 1980), an effect mediated by a major component of tunicamycin which inhibits the formation of GIcNAc pyrophosphoryl polyisoprenol (TAKATSUKI et al.. 1975: TKACZ and LAMPEN. 1975). The general effect of tunicamycin on total fat body glycoprotein biosynthesis was manifested in various complex ways in the synthesis of the storage proteins by treated tissues. Fat body from G. mellonella larvae at the stage of development used in these studies normally synthesizes four storage polypeptides having apparent subunit mol. wts of 74, 76, 81 and 82 K daltons (MILLERand SILHACEK,1982b). [mmunoprecipitation of the 74 and 76 K Mr storage proteins synthesized by treated tissues revealed the presence of two additional lower mol. wt variants within 2.5 hr of culture. Tissues exposed to tunicamycin for longer time periods (5 hr) synthesized only the two variants. and within 9 hr of culture even the synthesis of these had ceased, lmmunoprecipitation of the 81 and 82 K Mr polypeptides from homogenates of fat body

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~]EPHEN (i. MILLER and DONALD L. SII,HACEK

exposed to tunicamycin did not reveal the presence of tool. wt variants. The poor resolution of these two polypeptides on SDS gels, however, left open the possibility that the 82 K M r polypeptide was accumulating in the band position of the 81 K M, polypeptide. The absence of a band below 81 K was consistent with the observation that the latter has no detectable carbohydrate associated with it (MILLER and SILHACEK, 1982a). We were unable to demonstrate that the storage proteins synthesized by tunicamycin-treated fat body were non-glycosylated. The extremely low levels of storage proteins synthesized by treated fat body precluded binding analyses on lectin-derivatized agarose columns, one of the criteria used previously to determine the sugar content of the storage proteins (MILLER and SILHACEK,1982a). The presumption that these variants are not glycosylated is at least consistent with several independent observations including: {1) the 7,*, 76 and 82 K Mr storage proteins in G. mellonella contain mannose and GIcNAc (MILLER and SILHACEK, 1982a), carbohydrate constituents which are typically N-linked to asparaginyl residues (KORNEELD and KORNFELD, 1976); (2) the tunicamycinmediated reduction in apparent mol. wt of only 1-4 K (1.2-5.29o) is consistent with the 3.5~°,oby mass contributed by carbohydrate in the major storage protein of M . sexta (KRAMER et al., 1980); (3) both the synthesis of these variants and the inhibition of GIcNAc incorporation into fat body proteins occurred within 5 hr of exposure to tunicamycin; and (4) downward shifts in tool. wt have been documented for various nonglycosylated glycoproteins synthesized in the presence of tunicamycin, including human IgA (HICKMANet al., 1977), L cell interferon (FuJISAWAet al., 1978), yeast alkaline phosphatase (ONISVII et al,, 1979), chicken ovalbumin (STRUCK and LENNARZ, 1977) and the VSCG coat protein of trypanosomes (STRICKLERand PATTON, 1980). Exposure of G. mellonella fat body to tunicamycin also caused a rapid (within 5-9 hr) cessation of the export of proteins radiolabelled with both leucine and GIcNAc. Larval fat body normally exports approx. 20 newly synthesized polypeptides in vitro, the most prominent of which are the four storage proteins (MILLER and SILHACEK, 1982b). Immunoprecipitation of media in which treated tissues had been cultured revealed that a 2.5 hr exposure dramatically reduced, and a 5 hr exposure eliminated the export of all four storage proteins. Fluorographic analyses of the 74 and 76 K M~ polypeptides showed that only the putatively glycosylated forms of these proteins were exported following a 2.5 hr exposure, suggesting that carbohydrate is required for their release from fat body. Neither of these proteins was exported from fat body exposed to tunicamycin for 5 hr, in agreement with the observation that only the lower tool. wt variants are synthesized by fat body at this time. This interpretation of the role served by carbohydrate in mediating export is obscured somewhat by the concomitant reduction in the export of the 81 K M~ polypeptide by tissues treated with antibiotic over an identical culture period. This suggests that tunicamycin may also affect specific transport functions in fat body, an interpretation dictated by the fact that the normally non-glycosylated 81 K M~ polypeptide is

exported by untreated tissue (MILLERand SILt~ACEk. 1982b). These primary and secondary effects iS1Ru(k and LENNARZ, 1977: OLDEN et aL, 1979) of tunicamycin have been noted in other systems wherc certain non-glycosylated proteins fail to be exported {%x~ZER et al., 1977: H1CKMANet al., 1977: DUKSlN and BORr,STEIN, 1977) while others continue to be secrcted normally (OLDEN et al., 1978; LOH and GAINER, 1978: STRUCK et al., 1978). Tunicamycin also inhibited fat body protein synthesis but this effect was both less rapid (requiring approx. 9 hr of exposure) and less pronounced (maximum inhibition of 50°,; relative to controls) than its effect on glycoprotein biosynthesis. Analogous effects on protein synthesis mediated by tunicamycin have been noted (DuKSIN and BORNSTEIN, 1977: Tanzer et al., 1977; HICKMANet al., 1977: OLDEN et al., 1978). in G. mellonella fat body, at least part of this inhibition may be accounted for by rapid turnover of newly synthesized non-glycosylated storage proteins since these polypeptides were not detected in tissue treated with antibiotic for periods greater than 7 hr. These results provide indirect verification that insect glycoproteins are synthesized via a GlcNAcpyrophosphoryl-dolichol intermediate as they are in other animals (PARODI and LELOIR, 1979) and in higher plants (ELBEIN, 1979). Membrane preparations which transfer carbohydrate to an endogenous polyprenol acceptor have been characterized in both larval and pupal stages of Ceratitis capitata and Triatoma infestans (QUESADA ALLUE et at., 1975; BELOCOPITOW et al., 1977; QUESADA ALLUE and BELOCOPITOW, 1978). Insect fat body should prove to be ideally suited for studying both the mechanistic and regulatory aspects of glycoprotein biosynthesis, processing, intracellular transport, and secretion. Future analyses of G. mellonella fat body should be facilitated by the findings that: (1) large quantities of four proteins are secreted, and (2) carbohydrate contributes only a small percentage by mass in these proteins. Fat body from older larvae also resorbs these storage proteins from haemolymph (MILLER and SILHACEK, 1982b), and the role (if any) of carbohydrate in conferring specificity for this event also remains to be elucidated. REFERENCES BELOCOPITOWE., MARECHALL. R. and QUESADAALLUE L. A. (1977) Enzymatic synthesis of polyprenol monophosphate mannose in insects. Molec. Cell. Biochem. 16, 127-134.

DUKSINE. and BORSTEINP. (1977) Impaired conversion of procollagen to collagen by fibroblasts and bone treated with tunicamycin, an inhibitor of protein glycosylation. J. biol. Chem. 252, 955-962. ELBEINA. D. (1979) The role of lipid-linked saccharides in the biosynthesis of complex carbohydrates. A. Rev. Phmt Physiol. 30, 239-272. FUJISAWAJ., IWAKURAY. and KAWADEY. (1978) Nonglycosylated mouse L cell interferon produced by the action of tunicamycin. J. biol. Chem. 253, 8677-8679. GAHMBERG C. G., JOKINEN M., KARHI K. K. and ANDERSSON L. C. (1980) Effect of tunicamycin on the biosynthesis of the major human red cell sialoglycoproteim glycophorin A, in the leukemia cell line K562. J. biol. Chem 255, 2169-2175. HASILIKA. and TANNERW. 0978) Carbohydrate moiety of carboxypeptidase Y and perturbation of its biosynthesis. Eur. J. Biochem. 91,567-575.

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Fig. 3. Separation of [35S]-methionine-labelled proteins immunoprecipitated from homogenates of contro! fat bodies and those exposed to tunicamycin on SDS gels. Proteins precipitated with 74 K and 76 K-specific antiserum are shown, and each lane contained: (1) a mixutre of labelled, purified storage proteins as standards (18,600 dis/rain); (2) proteins from control tissues cultured for 7.5 hr (12.700dis/ min); (3) and (4) proteins from tissues exposed to tunicamycin for 2.5 (11,800 dis/min) and 5.0 (7800 dis/ mint hr, respectively. In all experiments the tissues were incubated with 20/~Ci/ml [35S]-methionine for the final 2.5 hr of culture. Dried gels prepared for fluorography were exposed to prefogged X-ray film for three days.

Fig. 4. Separation of [35S]-methionine labelled proteins immunoprecipitated from media in which control and tunicamycin-treated fat bodies had been cultured on SDS gels. Paired tissues were cultured in either 1 mi Grace's medium or medium containing 20 #g/ml tunicamycin for 2.5 or 5.0 hr, the final 2.5 hr of which was in the presence of 15 pCi/ml [35S]-methionine. Labelled exported proteins were precipitated with antisera to the 82 K Mr storage protein (lane (1): control, 14,000 dis/mis; lane (2): 2.5 hr tunicamycin exposure, 8400 dis/min; and lane (3): 5.0 hr exposure, 5200 dis/min) and antisera specific for the 76 and 74K Mr storage proteins (lane (4): control, 17,400dis/min; lane (5): 2.5 hr exposure to tunicamycin, 12,600 dis/rain; and lane (6): 5.0 hr exposure, 2700 dis/rain). Samples of total TCA~precipitable proteins from media in which control fat bodies had been cultured is shown in lane (6) (23,400 dis,.' min). Dried gels prepared for fluorography were exposed to prefogged X-ray film for three days.

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