Vol. 265, No. 12, ksue of April 25, pp. 686&&373, 1990 Printed in U.S.A.

Glycosylation of Nuclear Pore Protein ~62 RETICULOCYTE LYSATE CATALYZES O-LINKED N-ACETYLGLUCOSAMINE ADDITION IN VZTRO*

(Received for publication, December 5, 1989)

Christopher M. Starr* and John A. Hanover8 From the Laboratory of Biochemistry and Metabolism, National Znstitute of Diabetes, Digestive and Kidney Diseases, National Znstitutes of Health, Bethesda, Maryland 20892

The addition of O-linked N-acetylglucosamine (GlcNAc) to the major nuclear pore complex glycopro- tein p62 was examined. Expression of the rat p62 cDNA in transfected monkey cells was detected using a rat p62-specific antipeptide antiserum and two pre- viously described nuclear pore-specific monoclonal an- tibodies which require O-linked GlcNAc for binding. Although the p62 cDNA was predicted to encode a 54- kDa polypeptide, the product expressed in monkey cells migrated on sodium dodecyl sulfate-polyacryl- amide gel electrophoresis as two species of 62 and 59- kDa. Cell-free translation of the p62 z% uitro transcript yielded a 59-kDa polypeptide using wheat germ ex- tract and a 62-kDa product using a commercially avail- able rabbit reticulocyte lysate. Several lines of evi- dence indicated that the 62-kDa rabbit reticulocyte lysate translation product was modified by O-linked N-acetylglucosamine; the protein bound specifically to a wheat germ agglutinin affinity column and was con- verted to 59 kDa when treated with jack bean @-ace- tylglucosaminidase. The 59-kDa unglycosylated wheat germ translation product was converted to the 62-kDa glycosylated form upon incubation with reticulocyte lysate demonstrating that O-linked GlcNAc can be added to p62 post-translationally.

In addition to the well characterized N-linked and O-linked core glycosylation reactions which occur in the lumen of intracellular membranes, recent studies have revealed that glycosylation may also take place in the cytosol in the form of O-linked N-acetylglucosamine (GlcNAc) addition. Since the discovery of this novel saccharide modification, the list of proteins modified by O-linked GlcNAc has grown to include soluble cytoplasmic proteins, transcription factors, and cyto- skeletal proteins, as well as peripheral and integral membrane proteins at the cell surface and bound to intracellular mem- branes (Holt and Hart, 1986; Davis and Blobel, 1987; Holt et ul., 1987; Hanover et ul., 1987; Jackson and Tjian, 1988; King and Hounsell, 1989). These glycoproteins are all modified by single GlcNAc moieties attached directly to the peptide back- bone via 0-glycosidic linkages to serine (and possibly threo- nine) residues.

The nuclear pore complex contains a family of glycopro-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Held a National Research Council-National Institutes of Health/ National Institute of Diabetes, Digestive and Kidney Diseases Asso- ciateship while this work was done.

§ To whom all correspondence should be addressed. Tel.: 301-496- 0943.

teins, modiiied by O-linked GlcNAc, that range in molecular mass from 54,000 to 210,000 daltons. We have cloned the cDNA encoding the 62-kDa major nuclear pore glycoprotein, referred to as ~62, of the rat and determined the amino acid sequence of a site of O-linked GlcNAc addition (D’Onofrio et ul., 1988; Starr et ul., 1990). Our purpose in cloning p62 was to study the structure and glycosylation of the protein and to examine the role of p62 in mediating nucleocytoplasmic ex- change. Zn uitro experiments and studies in living cells suggest that the nuclear pore complex glycoproteins are involved in mediating the transport of macromolecules through the pore. Monoclonal antibodies prepared against nuclear pore glyco- proteins inhibit nuclear protein import and RNA efflux when microinjected into Xenopw oocytes (Dabauvalle et cd., 1988; Featherstone et ul., 1988). Reports that wheat germ agglutinin (WGA)’ binding blocks protein import both in uitro and in living cells suggest that the GlcNAc moieties of nuclear pore glycoproteins may be involved in the transport of proteins through the pore (Finlay et ul., 1987; Wolff et ul., 1988).

The biosynthesis of O-linked GlcNAc containing proteins is at present poorly understood. Davis and Blobel (1987) studies the timing of GlcNAc addition and reported the ab- sence of detectable unglycosylated p62 in labeled cell extracts suggesting that glycosylation occurs either co-translationally or within minutes of protein synthesis. Haltiwager et ul. (1990) used both a chemically deglycosylated acceptor protein and synthetic peptide acceptors for the detection of 0-glycosyl transferase activity in membrane fractions. The report of intrinsic membrane proteins with cytosolic facing O-linked GlcNAc is consistent with either the existence of a membrane bound 0-glycosyl transferase with an active site oriented toward the cytosol or a cytosolic transferase capable of acting on both soluble and membrane bound proteins (Capasso et al., 1988).

In this report, we described the addition of O-linked GlcNAc to the nuclear pore protein p62 in cells overexpressing rat p62 and during in uitro translation of p62 mRNA. These findings suggest that rabbit reticulocyte lysate contains the enzyme(s) required for transferring O-linked GlcNAc to p62 and that this glycosylation can occur post-translationally in the ab- sence of intracellular membranes.

MATERIALS AND METHODS

Transfection and Zmmunofluorescent Staining of COS-1 Cell-The rat p62 cDNA was constructed from clones isolated from an FRTL- 5 rat thyroid cell library and a library prepared by primer extension of rat liver mRNA as described previously (D’Onofrio et al., 198% Starr et aZ., 1990). The protein coding region of the rat p62 cDNA

’ The abbreviations used are: WGA, wheat germ agglutinin; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis; HEPES, 4-(2-hydroxyethylbl-piper- azineethanesulfonic aci&, NRK, normal rat kidney.

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was inserted downstream of the SV40 early promoter of pECE-SK (obtained from Frank McKeon, Harvard Medical School) to create the eukaryotic expression vector pECEc62. Monkey kidney COS-1 cells were transfected with uECEc62 using lipofectin (Bethesda Re- search Laboratories) at a ratio of 1 pg ofhpofectin/O.5 fig of DNA. The monoclonal antibody CHON-211 prepared against nuclear en- velope proteins of Chinese hamster ovary cells was described previ- ously (Park et ul., 1987). The monoclonal antibody RL-1, described by Snow et al. (1987), was a generous gift from Larry Gerace (Scripps Clinic). The rabbit anti-peptide serum designated AS474 was pre- pared against the synthetic peptide TTPSTSLPSLATQTS (Applied Biosystems, Foster City, CA) which corresponds to a sequence located in the amino terminus of rat p62 (Starr et al., 1990).

For immunofluorescence, cells grown on glass slides were fixed in 2% formaldehvde for 20 min. nermeabilized for 5 min in methanol at -20 ‘C, and incubated in primary antiserum diluted in PBS contain- ing 0.05% Tween-20 (TPBS) for 1 h at room temperature. The cells were rinsed three times in TPBS and incubated in the appropriate rhodamine (tetramethylrhodamine isothiocyanate) or fluorescein (fluorescein isothiocyanate) conjugated affinity purified secondary antibody (Jackson Immunologicals, West Grove, PA) for 30 min. Slides were mounted in fluid containing 90% glycerol, 10% PBS, and 0.1% 1*4-phenvlenediamine (Aldrich Chemical Co.) and photo- graphed using a Zeiss IM inverted fluorescence microscope equipped with a 100 X DlanaDOChrOmatiC obiective and Kodak Tri-X pan IS0 400/27’ print-film. -

Protein Analys&-Total protein homogenates from cultured cells grown in 35-mm tissue culture dishes were prepared by first washing adherent cells three times in PBS. Cells were then scraped into 0.5 ml of PBS precipitated in 2 volumes of 90% acetone for 30 min, and the protein pellet was resuspended in SDS-PAGE sample buffer. Rat liver nuclei were prepared according to the procedure described by Blobel and Potter (1966). SDS-PAGE was performed on a 9% acryl- amide:bisacrylamide (29:2:0.8) separating gel and blotted to nitrocel- lulose by the method of Towbin et ul. (1979). Protein blots were incubated with AS474 (1:2000) or with preimmune sera (1:2000). Immunoreactivity was detected using biotinylated goat anti-rabbit IgG and Vectastain AP (Vector Laboratories, Inc., Burlingame CA).

In Vitro Transcription and Cell-free 7Yan&&n-The protein cod- ing region of the rat p62 cDNA was subcloned downstream of the Sp6 RNA polymerase promoter in the transcription plasmid pGEM- 3Zf(+) (Promega, Madison, WI) to create the transcription vector pGEMc62. The pGEMc62 construct was transcribed in u&ro using Sp6 RNA polymerase (Promega). Guanylyltransferase (Bethesda Re- search Laboratories) was used to construct a G5-ppp5-N cap structure on the 5’ terminus of the p62 transcript product prior to in vitro translation according to the method described by Krieg and Melton (1984). The p62 transcript was translated in rabbit reticulocyte lysate (Bethesda Research Laboratories) or wheat germ extract (Promega) containing [“S]methionine (1000 Ci/mmol) (Amersham Corp.). Translation products were analyzed for carbohydrate content by digestion with 0.6 units of jack bean /3-hr-acetylglucosaminidase (Sigma) in 50 mM citrate-PO4 buffer, pH 4, containing 0.006 unit of aprotinin (Sigma), 1 pg of leupeptin (Sigma), and 1 fig of a-2 macroglobulin (Boehringer Mannheim) for 24 h at 37 OC. Translation products were also subj&ted to alkaline B-elimination using 0.5 M NaCl for 18 h at 24 ‘C. RNase A-treated wheat germ translation products were prepared by adding 1 ~1 of RNase A (1 mg/ml) (Be- thesda Research Laboratories) to i0 ~1 of the translation mixture for 5 min at 37 ‘C. Denaturing 9% SDS-PAGE gels of translation prod- ucts were dried and autoradiographed on Kodak X-Omat AR2 t?m.

Wheat Germ Agghtinin Affinity Chromatograp/ry-N-acetylglucos- aminyl-glycoprotein affinity columns were prepared using 2 ml of agarose-bound succinyl-WGA (binding capacity 2 mg of GlcNAc/ml of gel resin) (Vector Laboratories) in 0.5 M HEPES, pH 7.5, contain- ing 0.1 M NaCl. Aliquots of the translation mixture were applied to the column in HEPES-NaCl loading buffer. After extensive washing with loading buffer, the column was washed with 0.3 M glucose, pH 3.0, followed by elution of bound material with 0.3 M GlcNAc, pH 3.0. After aliquots were removed for liquid scintillation counting, acetone precipitates from the column fractions were dissolved in SDS-PAGE solubilization buffer and applied to a 9% SDS-PAGE gel.

RESULTS

Expression of Rat ~62 in Transfected Monkey &&-The rat p62 expression vector pECEc62 was introduced into COS-

1 cells by cationic liposome mediated transfection. Rat p62 expression was detected in these cells using the rabbit anti- peptide serum AS474 prepared against a synthetic peptide corresponding to a region near the amino terminus of rat ~62 (see “Materials and Methods”). Indirect immunofluorescence of transfected COS-1 cells (transCOS-1) using AS474 showed that rat p62 was present as phase-dense cytoplasmic aggre- gates in cells overexpressing the pore protein (Fig. 1, pane!.s b and e). Nonexpressing cells in the same fields did not react with AS474 demonstrating that the antiserum recognized rat p62 but not monkey nuclear pore proteins (see also Fig. 2). To confirm that the material detected by AS474 was indeed rat ~62, double-label immunofluorescence was performed using two previously characterized nuclear pore specific monoclonal antibodies. These monoclonal antibodies (RL-1 and CHON-211) are known to require the presence of O- linked GlcNAc for binding to the nuclear pore proteins and are not species specific (Snow et al., 1987, Park et al., 1987). Both CHON-211 (Fig. lc) and RL-1 (Fig. lf) were found to bind to the cytoplasmic aggregates detected by AS474 in Fig. 1, panels b and e, respectively. Nuclear envelope and punctate staining of the nuclei of both expressing and nonexpressing cells suggest that unlike AS474, the monoclonal antibodies also detect endogenous monkey nuclear pore proteins (Fig. 1, pane!s c and f). The ability of the monoclonal antibodies to recognize the cytoplasmic aggregates in transfected cells sug- gested that the rat p62 expressed in these monkey cells was modified by O-linked GlcNAc. In other double-label experi- ments, the cytoplasmic aggregates of rat p62 in transCOS-1 cells were found to bind fluorescently labeled wheat germ agglutinin (Starr et al., 1990). Taken together, these results implied that rat p62 synthesized in transCOS-1 cells con- tained O-linked GlcNAc and prompted us to examine the extent of glycosylation of rat p62 in these cells.

The open reading frame of the rat p62 cDNA encodes a protein with a calculated molecular weight of approximately 54 kDa (Starr et al., 1990). To determine the molecular mass of rat p62 expressed in transCOS-1 cells, total cell homoge- nates were subjected to SDS-PAGE followed by immunoblot- ting using AS474. AS474 recognized p62 as a single band at 62 kDa in isolated rat liver nuclei (RLN) (Fig. 2, lane 2) which was not detected by preimmune serum (Fig. 2, lane 1). AS474 also detected a 62-kDa species in NRK cells (NRK) (Fig. 2, lane 3). AS474 did not recognize proteins in nontrans- fected COS-1 cells (wtCOS-1) (Fig. 2, lane 5). However, in transCOS-1 cells, AS474 recognized two bands with molecular masses of approximately 59 and 62 kDa (Fig. 2, lane 4).

In Vitro Translation of p62 and Glycosylation by Rabbit Reticulocyte Lysate-In vitro transcription-translation was used to generate p62 for comparison with the rat p62 ex- pressed in transCOS-1 cells. The protein coding region of the rat p62 cDNA was inserted into the plasmid transcription vector pGEM-3Zf(+) and p62 mRNA was synthesized in vitro. The p62 mRNA was translated in cell-free systems containing either rabbit reticulocyte lysate or wheat germ extract. Using wheat germ extract, the p62 in vitro translation product migrated at approximately 59 kDa on SDS-PAGE (Fig. 2, lane 7). This polypeptide comigrated with the lower molecular weight band detected by AS474 in transCOS-1 cells (Fig. 2, lane 4). However, when the p62 mRNA was translated in rabbit reticulocyte lysate, a 62-kDa species was observed (Fig. 2, lane 6). No radiolabeled bands were detected on SDS- PAGE of cell-free translations performed without the addition of p62 mRNA (data not shown).

The larger size of the p62 in vitro translation product produced in rabbit reticulocyte lysate suggested that it might

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6S’iO Glycosylation of Nuclear Pore Glycoprotein ~62

FIG;. I. Expression ok p62 in transfected cells monitored by dou- ble-label indirect immunofluores- cence. Transfected COS-1 cells express- lng rat p62 were fIxed and prepared for indirect i~n~n~]~~olluorescence as de- scrlhed under “Methods and Materials.” The primary antibodies used were anti- peptide serum AS-474 and the mono- clonai antibodies CHON-21 1 and RLl. 1’on~/\ (1 and cf show the phase contrast images of transCOS-1 cells. These cells were double-labeled with AS474 (l:lOOO) (pin& b and <J) followed by lluorescein isothlocyanate-conjugated AP goat anti- rabbit IgG (1:20) and with either CHON- 21 1 (1:X)0) Cpr~nc/ c) or RI,-1 (1:5OO)(po~/ 1) followed by tetrameth- .vlrhodamine isothiocyanate-conjugated AI’ goat anti-mouse IgM (120). The cor- responding cartons in pon& b and c and the W~WL~ in ~WZ& e and f show that the cvtoplasmic aggregates stained by AS4’7i are recognized by both CHON- 211 and RLl. Note in pmd.~ b and c that cells not overexpressing the rat p62 (lower cell in each panel) show no signal with the species-specific anti-peptide serum AS474. However, the endogenous monkev nuclear pore proteins are de- tected jn these nonexpressing cells with hoth ot the monoclonal antibodies which react with nuclear pore glycoproteins (pon& c and /).

b

be modified by O-linked GlcNAc. Since only trace amounts of p62 were synthesized in the lysate during cell-free trans- lation, direct chemical confirmation of the presence of O- linked GlcNAc modification of p62 proved impractical; alka- line p-elimination resulted in complete degradation of the radiolabeled translation products synthesized in both reticu- locyte lysate and wheat germ extract (data not shown) (see “Materials and Methods”). However, several indirect methods were used which strongly suggested that the 62-kDa transla- tion product synthesized in reticulocyte lysate was modified by O-linked GlcNAc. The translation products were treated with b-N-acetylglucosaminidase and analyzed by SDS-PAGE. The mobility of the 59.kDa wheat germ translation product (Fig. 3, !ane 1) was not altered following treatment with B-N- acetylglucosaminidase (Fig. 3, lune 2). However, the rabbit reticulocyte lysate translation product, which migrated ap-

proximat.ely 3 kDa higher at 62 kDa (Fig. 3, lune 3) was found to comigrate with the 59-kDa wheat germ translation product following digestion with /3-N-acetylglucosaminidase (Fig. 3, lane 4).

The sensitivity of the 62-kDa reticulocyte lysate translation product to B-N-acetylglucosaminidase suggested it might con- tain covalently attached terminal GlcNAc. If this was the case, the material would be expected to bind to succinyl- WGA. Unlike native WGA that binds both sialic acid and GlcNAc moieties, succinyl-WGA demonstrates a unique bind- ing specificity for terminal GlcNAc residues (Monsigny et ul., 1980). When the reticulocyte lysate translation mixture was applied to a succinyl-WGA-agarose column a labeled species was retained on the lectin column that eluted with 0.3 M GlcNAc (Fig. 4, punel A). When analyzed by SDS-PAGE the material eluting from the column with GlcNAc was found to

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FIG. 2. Immunological detection of p62 expression in trans- fected cells and comparison with p62 in vitro translation products. On the left (larzes Z-5), samples were subjected to immu- noblotting as described under “Materials and Methods.” The anti- peptide antisera AS474 (1:2000 dilution) was used to monitor p62 expression in rat liver nuclei (RLN, lane 2), NRK cell homogenate (N&Y, lune 3), transCO81 cell homogenate (lcme 4), and nontrans- fected ( UJ~UXS-1) cell homogenate (lane 5). Rat liver nuclei incubated with rabbit preimmune sera (1:2000) is shown in lune 1. On the right (&es 6 and 7) is an autoradiogram showing the migration of [“S] methionine-labeled p62 translation products produced in rabbit retic- ulocyte lysate (RRL, lane 6) or wheat germ extract (WGX, lane 7) applied to the same SDS-PAGE gel. AS474 detects two bands in transCOS-I cells (lone 4) with migration patterns identical to the 59- kDa wheat germ translation product and the 62-kDa reticulocyte lysate translation product. Migration of molecular mass standards is shown on the left.

M. W. kDa

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Glycosylation of Nuclear Pore Glycoprotein p62 6871

be the 62-kDa translation product (Fig. 5, lane 2). However, when the 59-kDa wheat germ translation product was applied to the same lectin column, no radiolabeled material was eluted with 0.3 M GlcNAc (Fig. 4, panel B, and Fig. 5, lane 4).

Glycosylation of p62 Can Occur Post-translationally-It is not yet established whether O-linked GlcNAc addition is a co-translational or post-translational event. To determine whether O-linked GlcNAc could be added to p62 post-trans- lationally, the nonglycosylated 59-kDa wheat germ transla- tion product was incubated with rabbit reticulocyte lysate following the termination of protein synthesis as described under “Methods and Materials.” After incubation with the rabbit reticulocyte lysate for 5 min at 37 C, the wheat germ product acquired the ability to bind to succinyl-WGA and was specifically eluted from the affinity column with 0.3 M GlcNAc (Fig. 4, panel C). The material eluted from the column mi- grated as 62 kDa on SDS-PAGE (Fig. 5, lane 6). Therefore, as a result of the 5-min post-translational incubation with reticulocyte lysate, the wheat germ translation product shifted from 59 to 62 kDa and in the process acquired the ability to bind succinyl-WGA. This shift in apparent molecular weight is consistent with the post-translational addition of 8-10 GlcNAc residues to the wheat germ translation product. This finding suggests that the addition of O-linked GlcNAc to p62 can occur post-translationally.

DISCUSSION

The nuclear pore complex contains a family of proteins that are modified by O-linked GlcNAc; the major pore glyco- protein p62 has been estimated to contain 8-10 GlcNAc residues (Holt et al., 1987, Hanover et al., 1987, Davis and Blobel, 1987). The enzymatic requirements and intracellular location of the 0-glycosyltransferase(s) responsible for O- linked GlcNAc addition are not currently known. We have found that rat p62 synthesized in COS-1 cells transfected with the rat p62 cDNA binds to fluorescein conjugated WGA in situ (Starr et al., 1996). Consistent with this finding, rat p62 overexpressed in transCOS-1 cells was detected by two nuclear pore-specific monoclonal antibodies which require the presence of O-linked GlcNAc for binding. By SDS-PAGE, rat p62 synthesized in transfected cells is present as both a 59- kDa and 62-kDa species (Fig. 2, lane 4). These results suggest that a significant fraction of the p62 synthesized in transCOS- 1 cells contains O-linked GlcNAc.

1 2 3 4

FIG. 3. Sensitivity of in uitro translation products to B-N- acetylglucosaminidase. The p62 cDNA was transcribed in uitro using Sp6 polymerase. The p62 mRNA was translated in either wheat germ extract ( WGX, lane 1) or rabbit reticulocyte lysate (RRL, he 3). Translation products were treated with 0.6 unit of jack bean P-N- acetylglucosaminidase (GkNAcase) to remove O-linked GlcNAc. The 62-kDa translation product from the reticulocyte lysate translation (he 3) was reduced to 59 kDa following incubation with P-N- acetylglucosaminidase (lane 4). Incubation with @-N-acetylglucosa- minidase did not alter the size of the wheat germ extract translation product (compare lunes I and 2). Migration of molecular mass stand- ards is shown at Left.

Here we report that components present in commercially available rabbit reticulocyte lysate are sufficient for the ad- dition of O-linked GlcNAc to p62 in uitro. A number of lines of evidence suggest that rat p62 synthesized in membrane- depleted rabbit reticulocyte lysate is modified by O-linked GlcNAc. First, when treated with /3-N-acetylglucosaminidase, the 62-kDa reticulocyte translation product was converted to a 59-kDa species which comigrated on SDS-PAGE with non- glycosylated p62 synthesized using wheat germ extract. Fur- thermore, the 62-kDa reticulocyte lysate translation product bound specifically to a succinyl-WGA column, whereas the 59-kDa wheat germ translation product did not bind to suc- cinyl-WGA. Therefore, the cytosolic fraction of erythroid cells is a source of the glycosyl transferase responsible for O-linked GlcNAc addition. Since the reticulocyte lysate preparation used for cell-free translation is devoid of membranes, mem- brane association is apparently not required for the addition of O-linked GlcNAc to p62 in uitro.

Rat p62 synthesized using wheat germ extract migrates at 59 kDa on SDS-PAGE. Based solely on the molecular mass of individual GlcNAc residues, a shift in apparent molecular weight of 2200 would be expected for the addition of 10

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Glycosylation of Nuclear Pore Glycoprotein p62

10 20 30 0

Fraction Number FIG. 4. Succinyl-wheat germ agglutinin affinity chromatography of p62 in &ro translation prod-

ucts. p62 in uitro translation products were applied to a succinyl-WGA-agarose column, washed with 0.3 M glucose, and bound material was subsequently eluted with 0.3 M GlcNAc. Shown are the elution profiles of rabbit reticulocyte lysate translation products (panel A) and wheat germ extract translation products (punel R). The addition of glucose and GlcNAc are indicated by urrows. Radiolabeled material was eluted with GlcNAc from the reticulocyte lysate but not the wheat germ extract translations. To determine if the wheat germ translation product could be glycosylated post-translationally, the 59-kDa translation product was incubated with rabbit reticulocyte lysate (see “Materials and Methods”). As shown in pcznel C, the post-translationally glycosylated wheat germ translation product was retained on the succinyl-WGA column and eluted with 0.3 M GlcNAc. SDS-PAGE of material eluted from the columns with 0.3 M GlcNAc is shown in Fig. 5

FIG. 5. SDS-PAGE of p62 translation products bound and eluted from succinyl-WGA column. The translation products from the in uitro p62 transcript were applied to a succinyl-WGA column and eluted from the column with 0.3 M GlcNAc. The elution profile of each of the translation products is shown in Fig. 4. Paired lanes show the material applied to the column followed by the material that eluted with 0.3 M GlcNAc. Fractions 20, 21, and 22 from each column were pooled, acetone-precipitated, and applied to the gel. Lanes 1 and 2 show translation products synthesized using reticulocyte lysate: the 62-kDa translation product applied to the column (Lane I ) was eluted with 0.3 M GlcNAc (lane 2). The 59-kDa wheat germ translation product applied to the column (Lune 3) was not specifically eluted with 0.3 M GlcNAc (Lurze 4). However, when the 59-kDa wheat germ translation product was incubated with rabbit reticulocyte lysate, the apparent molecular mass increased to 62 kDa (compare lunes 3 and 5), which bound to the succinyl-WGA affinity column and eluted with 0.3 M GlcNAc (Lane 6).

residues to ~62. We have observed that the addition of O- linked GlcNAc moieties to the 59-kDa species of p62 increases its apparent molecular mass on SDS-PAGE by approximately 3 kDa. A similar shift in molecular mass was reported previ-

ously by Davis and Blobel (1987) using /3-IV-acetylglucosa- minidase to enzymatically deglycosylate p62 from isolated rat liver nuclei.

The data presented in this study is consistent with the post- translational addition of GlcNAc to ~62. Previously, Davis and Blobel (1987) examined the biosynthesis of p62 by pulse- chase experiments. They found that even at the earliest time point (0 chase) p62 was modified by O-linked GlcNAc and concluded that O-linked GlcNAc addition occurred either co- translationally or within 5 min from the start of translation. We have shown that the glycosyltransferase in reticulocyte lysate is capable of adding O-linked GlcNAc to nonglycosy- lated p62 post-translationally, although we cannot rule out the possibility that glycosylation may also occur co-transla- tionally in uiuo. The post-translational addition of O-linked GlcNAc is both rapid and quantitative with glycosylation essentially complete within 5 min resulting in a shift in molecular weight consistent with that observed in uivo. In COS-1 cells transfected with rat p62 cDNA, both the 62-kDa- glycosylated and 59-kDa-nonglycosylated forms of p62 are present. The fact that not all the rat p62 synthesized in these cells is glycosylated suggests that the glycosyltransferase ac- tivity may not be sufficient to glycosylate the large amount of rat p62 expressed in these transfected cells.

The demonstration of O-linked GlcNAc addition to GlcNAc during in uitro translation in a membrane-depleted reticulo- cyte lysate has several important implications. First, it can no longer be safely assumed that proteins translated in mem- brane depleted reticulocyte lysate are not glycosylated the translation products may be modified by O-linked GlcNAc. Second, the availability of radiochemically pure p62 will sim- plify identification and purification of the glycosyl transfer- ase(s) involved in the transfer of O-linked GlcNAc. Finally, the commercial availability of rabbit reticulocyte lysate pro- vides a convenient and practical means of catalyzing this recently identified post-translational modification.

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C M Starr and J A HanoverN-acetylglucosamine addition in vitro.

Glycosylation of nuclear pore protein p62. Reticulocyte lysate catalyzes O-linked

1990, 265:6868-6873.J. Biol. Chem. 

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