Proc. Nati. Acad. Sci. USA Vol. 82, pp. 6143-6147, September 1985 Cell Biology (Glu62,Ala30,Tyr8). serves as high-affinity substrate for tyrosylprotein sulfotransferase: A Golgi ( sulfation/protein sorting/chromaffin granules/tyrosine /tubulin) RAYMOND W. H. LEE AND WIELAND B. HUTTNER* Department of Neurochemistry, Max-Planck-Institute for Psychiatry, 8033 Martinsried, Federal Republic of Germany Communicated by Fritz Lipmann, May 10, 1985

ABSTRACT Tyrosylprotein sulfotransferase, the enzyme as substrate are as follows: (i) the only sulfatable hydroxyl catalyzing the sulfation of proteins on tyrosine residues, was group in Glu,Ala,Tyr is on tyrosine, in contrast to physio- characterized by using the acidic polymer containing tyrosine logical protein substrates, which may also contain sulfatable (Glu62,Ala3',Tyr8). (referred to as Glu,Ala,Tyr) as exogenous carbohydrate, , and residues; (ii) "protein" substrate. After subcellular fractionation ofa bovine Glu,Ala,Tyr is available in completely unsulfated form in adrenal medulla homogenate, tyrosylprotein sulfotransferase sufficient quantities for enzymological studies, whereas activity was found to be highest in fractions enriched in Golgi physiologically sulfated proteins purified from bi6logical membrane vesicles. Tyrosylprotein sulfotransferase required sources need to be desulfated before being used as sub- the presence of a nonionic detergent for sulfation of exogenous strates; (iii) Glu,Ala,Tyr is likely to contain tyrosine residues Glu,Ala,Tyr, indicating an orientation of the catalytic site of in the vicinity ofacidic residues. A comparison of the enzyme toward the Golgi lumen. Tyrosylprotein the amino acid sequences surrounding the sulfated tyrosine sulfotransferase was solubilized by Triton X-100, suggesting residues in gastrin (10), cholecystokinin (11), fibrinopeptide that the enzyme was tightly associated with the Golgi mem- B (12), and hirudin (13) shows that these sequences always brane, possibly as an integral membrane protein. The apparent contain acidic amino acids. Using Glu,Ala,Tyr as substrate, Golgi localization of tyrosylprotein sulfotransferase was sup- we characterized tyrosylprotein sulfotransferase from bovine ported by the observation that of proteins in adrenal medullary Golgi fractions. The present results have intact cells was blocked by monensin and was in line with been reported in abstract form (14). previous observations that all tyrosine-sulfated proteins known so far are secretory. Glu,Ala,Tyr was found to have a very high affinity for tyrosylprotein sulfotransferase (apparent Kmi 300 METHODS nM), similar to that reported for certain tyrosylprotein kinases. Cell Cultures. Cells were grown and harvested as described While this may suggest some similarity between these , (8). Homogenates were prepared by adding 4 vol of Hepes the Golgi localization of tyrosylprotein sulfotransferase segre- solution (10 mM Hepes, pH 7.3/5 mM 2-mercaptoethanol) to gates tyrosine sulfation from the sites of tyrosine phosphoryl- 1 vol of cell pellets, followed by 10 strokes at 3000 rpm in a ation of proteins in the intact cell. If, however, tyrosylprotein glass/Teflon homogenizer. Homogenates were centrifuged sulfotransferase was allowed to react with cytoplasmic proteins for 1 hr at 165,000 x g and aliquots of the resulting pellets by using a nonionic detergent, tyrosine sulfation of tubulin was (total membrane fraction) were used in the standard sulfation observed. reaction described below. Subcellular Frationation. Subcellular fractionation of adult Previous reports from this laboratory have established that bovine adrenal medulla was done essentially as described by sulfation of proteins on tyrosine residues is a ubiquitous Trifaro and Duerr (15), except that 5 mM 2-mercaptoethanol posttranslational modification in animal cells (1-6). Various was used throughout. Briefly, the homogenate was centri- lines of evidence have indicated that tyrosine-sulfated pro- fuged at 800 x g for 10 min, and the resulting postnuclear teins belong mostly, if not exclusively, to one topological supernatant was centrifuged at 20,000 x g for 20 min. The class: the secretory proteins (see refs. 2-7 and references resulting pellet, the crude granule fraction (15), was then therein). One important approach to understanding the role of subfractionated by discontinuous sucrose density gradient tyrosine sulfation ofsecretory proteins is the characterization centrifugation to obtain the Golgi-enriched fraction (0.8/1.0 of the enzyme(s) catalyzing this protein modification. We M sucrose interface) (15). The supernatant obtained after the previously described an enzymatic activity in rat pheochro- centrifugation at 20,000 x g was centrifuged at 114,000 x g that transferred sulfate for 70 min to yield the soluble fraction and the crude mocytoma (PC12) cell membranes to microsomal fraction, which was resuspended in sucrose tyrosine residues of proteins, and we designated this enzyme solution (0.3 M sucrose/5 mM 2-mercaptoethanol). Aliquots tyrosylprotein sulfotransferase (8). This enzyme catalyzed ofeach subcellular fraction were stored at -20°C. Galactosyl sulfation of the same proteins that were tyrosine-sulfated by transferase activity in the various subcellular fractions was intact cells (8). The cosubstrate for sulfation was 3'- measured as described (16). phosphoadenosine 5'-phosphosulfate (PAPS), the universal Solubilization of Tyrosylprotein Sulfotransferase. Aliquots sulfate donor discovered by Lipmann and colleagues (for ofthe Golgi-enriched fraction were subjected to the following review, see ref. 9). three treatments. (i) The Golgi-enriched fraction (1 ml, In this study, we used the acidic random polymer contain- corresponding to 1.4 mg of protein) was diluted with 9 ml of ing tyrosine, (Glu62,Ala30,Tyr8), (referred to as Glu,Ala,Tyr), Hepes solution and then centrifuged at 165,000 x g for 60 as exogenous "protein" substrate to characterize tyrosyl- min. The supernatant was concentrated to 1 ml by vacuum protein sulfotransferase. The reasons for choosing Glu,Ala,Tyr dialysis against Hepes solution. The pellet was resuspended in 1 ml ofHepes solution. (ii) The Golgi-enriched fraction was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: PAPS, 3'-phosphoadenosine 5'-phosphosulfate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6143 Downloaded by guest on September 26, 2021 6144 Cell Biology: Lee and Huttner Proc. Natl. Acad. Sci. USA 82 (1985) treated as in i, except it was diluted with Hepes solution accessibility of PAPS to sulfotransferases, all reactions were containing 150 mM NaCl, and this suspension, prior to performed in the presence of nonionic detergent. In the centrifugation, was sonicated four times for 15 sec on ice at primary subfractions (Fig. 2, lanes A-D), most of the endog- maximum power, using a Branson B 15 Sonifier. (iii) The enous sulfation was found in the crude granule fraction [Fig. Golgi-enriched fraction was treated as in i, except that after 2, lane B(-)]. Subfractionation of the crude granule fraction centrifugation the pellet was resuspended in Hepes solution on a discontinuous sucrose density gradient (Fig. 2, lanes with 30% (wt/vol) glycerol/1% (wt/vol) Triton X-100, to a E-K) showed that most of the endogenous sulfation was protein concentration of 3 mg/ml, kept on ice for 1 hr, and observed in the fraction collected at the 0.8/1.0 M sucrose centrifuged at 165,000 x g for 60 min. The supernatant was interface [Fig. 2, lane G(-)]. This fraction is known to be collected and the pellet was resuspended in Hepes solution enriched in Golgi membrane vesicles, as determined by containing glycerol and Triton X-100 to the volume of the assaying for galactosyl transferase (ref. 15 and Fig. 2 Lower), supernatant. Aliquots (25 1.l) of the supernatant and pellet a trans-Golgi marker (19). A small amount of endogenous from treatments i-iii were then assayed for tyrosylprotein sulfation with a pattern similar to that of the Golgi-enriched sulfotransferase activity as described below. fraction was observed in the fraction collected at the 0.3/0.8 Tyrosylprotein Sulfotransferase Assay. Prior to the assay, M sucrose interface [Fig. 2, lane F(-)]. Approximately 30% the ethanol in [35S]PAPS (New England Nuclear; 0.5-3 of the total 35SO4 incorporated into proteins of the Golgi- Ci/mmol; 1 Ci = 37 GBq) was evaporated under a stream of enriched fraction [Fig. 2, lane G(-)] was recovered as nitrogen. The standard reaction mixture [see Fig. 2, lane tyrosine 35S4. The remaining 70% was alkali labile and was, G(+)] of 100 pl was composed of, in final concentrations, 10 therefore, presumably carbohydrate 35SO4. A different pat- mM Hepes, pH 7.0/5 mM MgCl2/5 mM MnCl2/5 mM tern of endogenous sulfation was observed in the fractions of 2-mercaptoethanol/1 mM NaF/20 1M [35S]PAPS/1% Triton the sucrose gradient enriched in chromaffin granules [Fig. 2, X-100/4 ,M Glu,Ala,Tyr (Sigma, P 3899; average size, 25 lanes J(-) and K(-)]. More than 97% of this incorporated kDa) and 70 ul of Golgi-enriched fraction (0.7-1.5 mg of 35 O4 appeared to be linked to carbohydrate residues. No protein per ml), which was added last to start the reaction. significant endogenous sulfation was observed in fractions The reaction mixture was incubated at 30°C for 25 min. This containing mitochondria [Fig. 2, lane H(-)] and lysosomes standard assay was modified as described in the figure [Fig. 2, lane I(-)], even when the lysosome fraction was legends. All reactions were terminated by adding 50 ,A of 3 assayed at pH 5.0 (data not shown). times concentrated NaDodSO4 stop solution; samples were The various fractions of the sucrose density gradient were analyzed by NaDodSO4/PAGE; and gels were stained, assayed for tyrosylprotein sulfotransferase activity toward destained, dried, and fluorographed as described (2, 8). Dried Glu,Ala,Tyr. [Another synthetic polymer, (Glu83, Tyr'7)n, gel pieces containing 35SO4-labeled Glu,Ala,Tyr were ana- was also found to serve as substrate, but with a lower lyzed for tyrosine 35S04 as described (2, 8). efficiency than Glu,Ala,Tyr (data not shown). Therefore, only Glu,Ala,Tyr was used routinely.] NaDodSO4/PAGE of RESULTS radioiodinated or chemically 35S04-labeled Glu,Ala,Tyr in- dicated that this random polymer migrated as a smear, Monensin Inhibits Tyrosine Sulfation of Proteins in Intact extending from a position corresponding to -50 kDa to the Cells. Fig. 1 shows that in intact rat pheochromocytoma cells dye front (data not shown). Migration of Glu,Ala,Tyr as a (PC12 cells), 0.1 uM monensin, which is known to inhibit smear on NaDodSO4/PAGE has also been observed when Golgi-associated functions (17), completely blocked the the polymer was phosphorylated by tyrosine kinases (20). 35SO4 incorporation into macromolecules, including that into When Glu,Ala,Tyr was added to the various fractions of the secretogranins I and II (18) [previously referred to as sucrose gradient [Fig. 2, (+) lanes E-K], a 35S04-labeled p113/plO5 and p86/p84, respectively (8)], the major tyrosine- smear in the characteristic position of Glu,Ala,Tyr was most sulfated proteins of these cells (8). This suggested that prominently seen in the Golgi-enriched fraction [Fig. 2, lane tyrosine sulfation of proteins may occur in the Golgi com- G(+), arrows]. At least 70% of the radioactive sulfate in the plex. Glu,Ala,Tyr region was recovered as tyrosine 35S04. In the Tyrosylprotein Sulfotransferase Activity Is Highest in the absence of Glu,Ala,Tyr, 1/6th as much tyrosine 35S04 was Golgi-Enriched Fraction of Adrenal Medulla. The sulfation of found in the same region of the gel [Fig. 2, lane G(-)]. The tyrosine and carbohydrate residues of endogenous substrate distribution of tyrosylprotein sulfotransferase in the sucrose proteins by endogenous sulfotransferases, referred to as gradient was similar to that of galactosyl transferase when endogenous sulfation (8), was determined in various expressed as both total activity and specific activity (Fig. 2 subcellular fractions [Fig. 2, (-) lanes]. To maximize the Lower). The specific activities of tyrosylprotein sulfo- transferase and galactosyl transferase in the Golgi fraction were enriched 6.8- and 6.2-fold, respectively, compared with A B the crude granule fraction; 48% and 55%, respectively, of the total activities of the two enzymes recovered from the found in the Golgi fraction. Ib FIG. 1. Tyrosine sulfation of pro- sucrose gradient were No 97 teins in intact PC12 cells is blocked by significant tyrosylprotein sulfotransferase activity was ob- II -W monensin. Cells were labeled for 3 hr served in the chromaffin granule fraction at pH 7.3 [Fig. 2, 67 with inorganic 31SO4 as described (8), in lanes J(+) and K(+)] or at pH 5.0 (data not shown). In the absence (-) or presence (+) of 0.1 addition to serving as substrate for tyrosylprotein sulfotrans- 43 ,uM monensin that was added to the ferase, Glu,Ala,Tyr caused an inhibition of endogenous a medium 1 hr before the isotope. La- sulfation of both tyrosine and carbohydrate residues in all 0 beled cells were analyzed by NaDod- fractions of the sucrose gradient and in the primary subfrac- 30 1 S04/PAGE. A fluorogram of the gel is tions [Fig. 2, (+) lanes]. This phenomenon was not investi- shown. Arrows indicate positions of the study. major tyrosine-sulfated PC12 cell pro- gated further in present 20 teins-secretogranin I (p113/plO5; ref. Orientation of the Catalytic Site of Tyrosylprotein Sulfotransferase Toward the Golgi Lumen. In the experiments bpb 8) and secretogranin II (p86/84; ref. 8). Positions of molecular size standards illustrated in Fig. 2, endogenous sulfation and sulfation in the are shown on the left; bpb, bromphenol presence of Glu,Ala,Tyr had been performed in the presence Monensin - + blue front. of the nonionic detergent Triton X-100. As shown in Fig. 3, Downloaded by guest on September 26, 2021 Cell Biology: Lee and Huttner Proc. Natl. Acad. Sci. USA 82 (1985) 6145 A B C D E F G H I K r- m m r ml m -l m --w qm-

.f. 97 a u _ 67 . a 43 30 F II 20 L

EAY: - + - + - + -+ - + - + - - + - + -

Total Activity Specific Activity

(noaIn10 10 0

'(D 8 8- L..- 75 6- 6-

(A. 4-

U,0 2- K/

0

0 f.CM E FOH I J K E F GH IlJK FIG. 2. Tyrosylprotein sulfotransferase activity is highest in subcellular fractions enriched in Golgi membrane vesicles. Subcellular fractions of bovine adrenal medulla homogenate were prepared as described in ref. 15 and in Methods. Lanes A-D: primary fractions; A, postnuclear supernatant; B, crude granule fraction; C, crude microsomal fraction; D, soluble fraction; lanes E-K: subfractionation of crude granule fraction on a discontinuous sucrose gradient; E, 0.3 M sucrose layer; F, 0.3 M/0.8 M interface; G, 0.8 M/1.0 M interface (Golgi-enriched fraction); H, 1.0 M/1.2 M interface (mitochondria); I, 1.2 M/1.4 M interface (mitochondria and lysosomes); J, 1.4 M/1.6 M interface including 1.6 M sucrose layer (lysosomes and chromaffin granules); K, pellet (chromaffin granules). In the standard sulfation reaction, 50 Ag of protein from each fraction was used in the absence (-) or presence (+) of Glu,Ala,Tyr (EAY). Proteins were analyzed by NaDodSO4/PAGE, followed by fluorography. The positions of molecular size standards are shown on the left. In lane G(+), the area between the arrows (including the dye front) represents the region of the gel containing 35S04-labeled Glu,Ala,Tyr. The radioactivity found in the Glu,Ala,Tyr region in lanes E-K was analyzed for the presence of tyrosine "SO4. (Lower) Total activity and specific activity of tyrosylprotein sulfotransferase is expressed as pmol of tyrosine 3"S04/min per total subcellular fraction and as pmol x 10-1 oftyrosine 3"S04/min per mg of protein, respectively. Galactosyl transferase activity was also determined in fractions E-K and is represented by shaded bars. Total activity and specific activity ofgalactosyl transferase is expressed as nmol x 10-1 of [14C]galactosyl-ovomucoid/min per total subcellular fraction and as nmol x 10-1 of [14C]galactosyl-ovomucoid/min per mg of protein, respectively.

sulfation of Glu,Ala,Tyr was only observed if the Golgi activity toward Glu,Ala,Tyr (Fig. 4 Right, lane I) and of the membrane vesicles had been dissolved by the detergent (Fig. endogenous sulfation activity (data not shown) remained 3, lane D). Likewise, the inhibitory effect of Glu,Ala,Tyr on associated with the Golgi membrane. The same was observed endogenous sulfation was seen only in the presence of with sonication in the presence of 150 mM NaCl (Fig. 4 Right, detergent. In contrast, endogenous sulfation of the Golgi- lane II). When Golgi membrane vesicles were treated with enriched fraction was observed in both the absence (Fig. 3, 1% Triton X-100, -50% of the original tyrosylprotein lane A) and presence (Fig. 3, lane C) of the detergent. sulfotransferase activity and most of the endogenous sulfa- However, addition of detergent affected the pattern of tion activity was solubilized (Fig. 4 Left, lanes A-C; Right, endogenous sulfation, resulting in the appearance of three lane III). The reason for the incomplete solubilization of prominently sulfated bands of -58, -56, and -54 kDa (Fig. tyrosylprotein sulfotransferase is not known. 3, lane C). Whenl sulfation of the Golgi-enriched fraction was Properties of Tyrosylprotein Sulfotransferase. By using carried out in the presence of Triton X-100 and of added Glu,Ala,Tyr as substrate, some of the properties of purified bovine brain tubulin, the amount of sulfated 56-kDa tyrosylprotein sulfotransferase, as observed in Triton X-100- protein was markedly increased (Fig. 3, lane F) and the solubilized Golgi membrane vesicles, were investigated (Fig. sulfated 56-kDa protein was found to comigrate with the 5). Under standard conditions, sulfation of Glu,Ala,Tyr was added tubulin. This indicated that the sulfated 56-kDa band linear up to -30 min (Fig. SA). 35S04 incorporated into corresponded to sulfated tubulin. Approximately 70% of the Glu,Ala,Tyr after 30 min was not removed from Glu,Ala,Tyr radioactive sulfate incorporated into tubulin was recovered upon further incubation in the presence of excess unlabeled as tyrosine sulfate. PAPS, indicating the lack of a desulfating activity acting on Solubilization of Tyrosylprotein Sulfotransferase from Golgi Glu,Ala,Tyr under the present experimental conditions. We Membrane Vesicles. The nature of the association of have noticed that the efficiency of Glu,Ala,Tyr as substrate tyrosylprotein sulfotransferase with the Golgi membrane depended to some extent on the batch of GluAla,Tyr vesicles was investigated (Fig. 4). In low ionic strength obtained. In different experiments, 0.5-2.5 mol of sulfate was medium, almost all of the tyrosylprotein sulfotransferase incorporated into 100 mol of Glu,Ala,Tyr. Tyrosylprotein Downloaded by guest on September 26, 2021 6146 Cell Biology: Lee and Huttner Proc. Natl. Acad. Sci. USA 82 (1985)

A B C D E F

2:. 05°

x 5 E 67 67 L43 C 0 3~~~~~~~4 Time, min NaCI, M pH =30 s + 30 0.8 ca D 1.0- E 20. 2 -C O6 / 00.8- 0.6- 4 20 2 0O4- 2 EAY - - | - Tubulin 0.2 Triton X-100 - - + + + E 5 10 10 -0.51 0 0.! FIG. 3. Catalytic site for tyrosylprotein sulfotransferase is ori- 0 2 4 6 8 10 0 5 10 15 20 ented toward the luminal side of Golgi membrane vesicles. The GluAlaTyr, pM PAPS, ,LM Golgi-enriched fraction (100 ,ug of protein) was sulfated using the FIG. 5. Properties of tyrosylprotein sulfotransferase in Golgi- standard assay condition, in the absence (-) or presence (+) of4 ,AM enriched fraction. (A) Solid line, standard reaction mixture contain- Glu,Ala,Tyr (EAY), 130 ,ug of purified bovine tubulin per ml, or 1% ing 33 ,ug of protein and 5 Glu,Ala,Tyr was incubated for various Triton X-100, as indicated. Lanes A-D and E and F are from two ,uM times at 370C; dashed lines, standard reaction mixture 100 different experiments. Fluorograms of the gels are shown. Arrows containing ,ug of protein and 4 Glu,Ala,Tyr was incubated for 30 min at 300C indicate positions of tubulin, as determined by Coomassie blue AuM staining. with (A) or without (v) MgCl2 and MnCl2; after 30 min in the presence of MgCl2 and MnC12, reaction continued with (A) or without (v) 500 ,uM unlabeled PAPS. (B) Standard reaction mixture containing as the sulfotransferase was inactive when both MgCl2 and MnCl2 source of enzyme the Triton X-100 supernatant ofthe Golgi-enriched were omitted (Fig. 5A). MgCl2 (5 mM) or MnCl2 (5 mM) alone fraction (see Fig. 4) was incubated in the presence of various was found to be sufficient for sulfation (data not shown). concentrations of NaCl. (C) Standard reaction mixture without the Tyrosylprotein sulfotransferase activity was inhibited by Hepes buffer was incubated at various pH values, using 10 mM increased ionic strength ofthe reaction mixture; in particular, Tris-HCl/10 mM 2-(N-morpholino)ethanesulfonic acid titrated with at values >200- mM NaCl (Fig. 5B). The pH optimum for either HCl or NaOH. (A-C) All reactions were analyzed by NaDod- The into the of tyrosylprotein sulfotransferase was found to be between pH S04/PAGE. 35SO4 incorporation Glu,Ala,Tyr region the gels is shown. (D and E) Standard reaction mixture was incubated 5.3 and with a at 6.5 The of 7.0, peak pH (Fig. SC). affinity with various concentrations of either Glu,Ala,Tyr (D) or radioactive tyrosylprotein sulfotransferase for Glu,Ala,Tyr was found to PAPS (E). All reactions were analyzed by NaDodSO4/PAGE fol- be very high (Fig. SD). Lineweaver-Burk analysis indicated lowed by determination of the amount of tyrosine 31SO4 in the an apparent Km value of -0.3 ,uM Glu,Ala,Tyr, correspond- Glu,Ala,Tyr region of the gel. Tyrosylprotein sulfotransferase ac- ing to 7.5 /,g ofGlu,Ala,Tyr per ml. When calculated in terms tivity is expressed as shown on the ordinate of D. (Insets) of tyrosine concentration rather than Glu,Ala,Tyr concen- Lineweaver-Burk analysis of the results is shown. Apparent Km for 0.3 Km 5 tration, the apparent Km value was 5 ,uM. The apparent Km Glu,Ala,Tyr, ,uM; apparent for PAPS, ,uM. Similar values were obtained with 5-min and 10-min reactions. oftyrosylprotein sulfotransferase for PAPS was calculated to be S ,uM (Fig. SE). Tyrosylprotein Sulfotransferase Using Glu,Ala,Tyr as Sub-

A B C strate Is Present in Total Membrane Fractions ofVarious Cells. Three different cell lines, mouse, fibroblasts (L cells), rat _ 00 pheochromocytoma cells (PC12 cells), and mouse pituitary tumor cells (AtT-20 cells), were tested for the presence of 97- 50 IF tyrosylprotein sulfotransferase activity toward Glu,Ala,Tyr. 67 In a total membrane fraction prepared from these cells, added v40 43 Glu,AlaTyr became sulfated and inhibited the sulfation of endogenous substrate proteins, notably tubulin (data not ox X @ o 30 0 0 shown). 30- 020 DISCUSSION 20- 10 I The Golgi complex has been shown to be the major L subcellular compartment where carbohydrate residues of EAY: -+ -+ -+ I 11I HI1 glycoproteins and proteoglycans become sulfated (for re- view, see ref. 21). Our results suggest that the sulfation of FIG. 4. Tyrosylprotein sulfotransferase is tightly associated with proteins on tyrosine residues also occurs in the Golgi com- the Golgi membrane. The Golgi-enriched fraction was subjected to plex. First, as shown by subcellular fractionation of the three different types of extraction, using either low ionic strength adrenal medulla, tyrosylprotein sulfotransferase activity was (treatment I), 150 mnM NaCl and sonication (treatment II), or Triton highest in Golgi-enriched membrane fractions and was vir- X-100 (treatment III). Lanes A-C show results of Triton X-100 tually absent from the chromaffin granule fraction. Second, extraction; lane A, pellet; lane B, supernatant; lane C, starting monensin blocked tyrosine sulfation of proteins in intact material used for extraction. Absence (-) and presence (+) of cells. Third, preliminary pulse-chase experiments with IgM Glu,Ala,Tyr (EAY) is indicated. Fluorograms of the gel are shown. "5s radioactivity in the Glu,Ala,Tyr regions of the gels was analyzed (unpublished data) indicate that sulfate is added to tyrosine for the presence of tyrosine 05SO4. Results of treatments I-III are residues at about the same time as galactose is added to shown in the bar graph. Tyrosylprotein sulfotransferase activity in N-linked oligosaccharides-i.e., in the trans-Golgi cisternae. the supernatant fractions is expressed as percentage of total recov- Fourth, tyrosine sulfation of has been reported to ered activity (sum of activity in supernatant and pellet). occur after the protein has left the rough endoplasmic Downloaded by guest on September 26, 2021 Cell Biology: Lee and Huttner Proc. Natl. Acad. Sci. USA 82 (1985) 6147 reticulum (22). However, we cannot exclude the possibility this study) and by tyrosylprotein kinases (20, 24, 25). While that tyrosine sulfation occurs in membrane vesicles shortly our study was in progress (see refs. 14 and 26), Braun et al. after proteins have left trans-Golgi cisternae, ifthese vesicles (20) reported that Glu,Ala,Tyr also serves as high-affinity would fractionate similarly to Golgi membrane vesicles. substrate for tyrosylprotein kinases. Furthermore, when the Tyrosylprotein sulfotransferase appeared to be tightly barrier of cellular membranes is bypassed unphysiologically, associated with the Golgi membrane, its catalytic site being secretory proteins known to become sulfated on tyrosine oriented toward the Golgi lumen. This localization of residues such as gastrin (10) can become phosphorylated (27), tyrosylprotein sulfotransferase in the Golgi complex is con- and cytoplasmic proteins with appropriate acidic tyrosine- sistent with the presence of a transmembrane carrier system containing sequences such as tubulin (28) can become sul- for PAPS in Golgi vesicles (23) and with the observation that fated on tyrosine (Fig. 3). It will be of interest to investigate, all in vivo tyrosine-sulfated proteins identified so far are with purified tyrosylprotein sulfotransferase, the cellular secretory (refs. 2-7 and references therein). effects of a "false" modification by sulfation of a physiolog- The biological significance of tyrosine sulfation is unclear. ically tyrosine-phosphorylated protein. We have suggested (3-5) that the occurrence of tyrosine sulfate in many secretory proteins, in view of the diverse We thank Prof. H. Thoenen for his continuous support; P. A. postsecretion properties of these proteins, may indicate a Baeuerle for synthesizing "SO4-labeled Glu,Ala,Tyr; Dr. E. function for this modification in a presecretion event-e.g., Friederich for L cells; J. Hesse and Dr. G. Isenberg for purified in the sorting of these proteins. This hypothesis implies an tubulin; A. Hille for AtT-20 and PC12 cells; P. A. Baeuerle, A. Hille, Dr. P. Rosa, Dr. M. Schleicher, and Dr. S. Saadat for their helpful important role for tyrosylprotein sulfotransferase in the comments on the manuscript; and H. Macher for typing the manu- control of secretion of certain proteins. How, then, is the script. W.B.H. was recipient of a grant from the Deutsche specificity of this enzyme for its physiological substrate Forschungsgemeinschaft (Hu 275/3-2). proteins achieved? In line with the considerations outlined in the Introduction, our results indicate that the presence of 1. Huttner, W. B. (1982) Nature (London) 299, 273-276. acidic amino acids near the tyrosine that becomes sulfated 2. Huttner, W. B. (1984) Methods Enzymol. 107, 200-223. appears to be one factor in the recognition of appropriate 3. Hille, A., Rosa, P. & Huttner, W. B. (1984) FEBS Lett. 177, substrate proteins by tyrosylprotein sulfotransferase. The 129-134. finding that Glu,Ala,Tyr served as high-affinity substrate for 4. Baeuerle, P. A. & Huttner, W. B. (1984) EMBO J. 3, the enzyme indicates that an appropriate arrangement of 2209-2215. 5. Rosa, P., Fumagalli, G., Zanini, A. & Huttner, W. B. (1985) J. , alanine, and tyrosine is sufficient for recogni- Cell Biol. 100, 928-937. tion by the enzyme. The observation that the stoichiometry 6. Baeuerle, P. A. & Huttner, W. B. (1985) J. Biol. Chem. 260, of Glu,Ala,Tyr sulfation was low suggests that the sequence 6434-6439. recognized by the enzyme was specific, occurring in only a 7. Liu, M.-C. & Lipmann, F. (1985) Proc. Natl. Acad. Sci. USA few percent of the Glu,Ala,Tyr molecules. 82, 34-37. We have recently observed (unpublished data) that the 8. Lee, R. W. H. & Huttner, W. B. (1983) J. Biol. Chem. 258, tyrosylprotein sulfotransferase of the Golgi-enriched fraction 11326-11334. catalyzes the sulfation of purified secretogranins, the major 9. Lipmann, F. (1958) Science 128, 575-580. tyrosine-sulfated proteins found in secretory granules of 10. Gregory, H., Hardy, P. M., Jones, D. S., Kenner, G. W. & Sheppard, R. C. (1964) Nature (London) 204, 931-933. various endocrine cells including chromaffin granules (18), if 11. Jorpes, J. E. & Mutt, V. (1973) in Handbook ofExperimental these proteins had been desulfated. This indicates that the (Springer, Berlin), Vol. 24, pp. 1-179. enzyme characterized in the present study with Glu,Ala,Tyr 12. Dayhoff, M. 0. (1972) in Atlas of Protein Sequence and as exogenous substrate indeed sulfates physiologically rele- Structure (National Biomedical Research Foundation, Silver vant proteins. It will be important to determine whether Spring, MD), Vol. 5, pp. D87-D97. tyrosylprotein sulfotransferase from adrenal medulla is sim- 13. Petersen, T. E., Roberts, H. R., Sottrup-Jensen, L., Staffan, ilar to the enzyme(s) catalyzing the sulfation of other phys- M. & Bagdy, D. (1976) in Protides of Biological Fluids, ed. iologically relevant proteins (e.g., gastrin and cholecystokin- Peeters, H. (Pergamon, New York), Vol. 23, pp. 145-149. in). 14. Lee, R. W. H. & Huttner, W. B. (1984) J. Cell Biol. 99, 231a(abstr.). We have suggested (1) that if tyrosine sulfation of proteins 15. Trifar6, J. M. & Duerr, A. C. (1976) Biochim. Biophys. Acta were found to occur in the same subcellular compartment as 421, 153-167. tyrosine phosphorylation of proteins, the balance of cellular 16. Bretz, R. & Staeubli, W. (1977) Eur. J. Biochem. 77, 181-192. regulation may be perturbed by "false" protein modification 17. Tartakoff, A. M. (1983) Cell 32, 1026-1028. because of the resemblance of sulfated and phosphorylated 18. Rosa, P., Hille, A., Zanini, A., Navone, F., De Camilli, P. & residues. Here, we provide two important points of informa- Huttner, W. B. (1985) J. Neurochem. 44, Suppl., S90A (abstr.). tion regarding that suggestion. First, in the cellular systems 19. Roth, J. & Berger, E. C. (1982) J. Cell Biol. 92, 223-229. studied so far, tyrosine sulfation and tyrosine phosphoryl- 20. Braun, S., Raymond, W. E. & Racker, E. (1984) J. Biol. ation of proteins appear to be within the Chem. 259, 2051-2054. strictly segregated 21. Farquhar, M. G. & Palade, G. E. (1981) J. Cell Biol. 91, cell, with tyrosine sulfation occurring on the luminal side of 77s-103s. Golgi membranes (Fig. 3) and tyrosine phosphorylation 22. Kudryk, B., Okada, M., Redman, C. M. & Blombaeck, B. occurring in the cytoplasm and the nucleus or on the (1982) Eur. J. Biochem. 125, 673-682. cytoplasmic side of membranes (for review, see ref. 24). 23. Schwarz, J. K., Capasso, J. M. & Hirschberg, C. B. (1984) J. Consistent with this, our results show that Glu,Ala,Tyr, Biol. Chem. 259, 3554-3559. when added to the cytoplasmic side of the Golgi vesicles, and 24. Sefton, B. M. & Hunter, T. (1984) Adv. Cyclic Nucleotide mem- Protein Phosphoryl. Res. 18, 195-226. tubulin, associated with the cytoplasmic side of Golgi 25. Rees-Jones, R. W., Hendricks, A., Quarum, M. & Roth, J. branes, did not serve as substrates unless the Golgi mem- (1984) J. Biol. Chem. 259, 3470-3474. branes had been dissolved by nonionic detergent. Second, it 26. Lee, R. W. H., Suchanek, C. & Huttner, W. B. (1984) J. Biol. is of interest that, irrespective of the separation of tyrosine Chem. 259, 11153-11156. sulfation and tyrosine phosphorylation from one another by 27. Baldwin, G. 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