©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Golgi Localization in Yeast Is Mediated by the Membrane Anchor Region of Rat Liver Sialyltransferase (*)

(Received for publication, July 7, 1994; and in revised form, November 28, 1994)

Tilo Schwientek Claudia Lorenz Joachim F. Ernst (§)

From the Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To investigate the function of the membrane anchor region of a mammalian glycosyltransferase in yeast we constructed a fusion gene that encodes the 34 amino-terminal residues of rat liver beta-galactoside alpha-2,6-sialyltransferase (EC 2.4.99.1) (ST) fused to the mature form of yeast invertase. Transformants of Saccharomyces cerevisiae expressing the fusion gene produced an intracellular heterogeneously N-glycosylated fusion protein of intermediate molecular weight between the core and fully extended N-glycosylated form of invertase, suggesting a post-endoplasmic reticulum (ER) localization. In two types of cell fractionation using sucrose density gradients the ST-invertase fusion protein cofractionated with Golgi marker proteins, whereas a minor fraction (about 30%) comigrated with a vacuolar marker; ST-invertase was not detected in other cell fractions including the ER and the plasma membrane. Consistent with Golgi localization, about 70% of the total amount of the ST-invertase fusion was immunoprecipitated with an antibody directed against alpha-1,6-mannose linkages. The results demonstrate that the membrane anchor region of a mammalian type II glycosyltransferase is able to target a protein to the secretory pathway and to a Golgi compartment of the yeast S. cerevisiae, indicating conservation of targeting mechanisms between higher and lower eukaryotes. Since typical yeast Golgi localization signals are missing in the ST-membrane anchor region the results also suggest that yeast as mammalian cells utilize diverse mechanisms to direct proteins to the Golgi.


INTRODUCTION

The Golgi comprises cellular compartments that secreted proteins traverse between the endoplasmic reticulum (ER) (^1)and their final destinations(1) . Individual Golgi compartments are defined by specific protein processing functions, especially by reactions leading to the trimming and extension of glycosyl chains(2) . In mammalian cells, a wide variety of glycosyl chains consisting of different sugars and glycosidic linkages are successively synthesized in neighboring cisternae of typical Golgi stacks(3) . Terminal galactosyl and sialic acid residues of complex glycosyl chains are added, respectively, in the trans-Golgi or the trans-Golgi network compartments. In the yeast Saccharomyces cerevisiae the Golgi does not need to carry out as diverse functions as its mammalian counterpart, since yeast glycosyl structures are relatively simple(4) . It is known that the addition of mannosyl residues to N- and O-linked glycosyl chains, as well as the processing of the precursor of the alpha-factor pheromone and killer factor occur in the yeast Golgi(5) . The yeast Golgi is subdivided in an early compartment containing alpha-1,6-mannosyltransferase, a medial compartment containing Man-Tf and GDPase and a late compartment containing processing proteases Kex2p and Ste13p(6, 7) . A rudimentary stack of Golgi cisternae is visible only in certain secretory mutants of S. cerevisiae(8) .

The various Golgi processes require membrane proteins that are localized in specific Golgi compartments. The common structure of these enzymes consists of a cytoplasmic tail, a membrane anchor region, and a luminal region required for enzymatic function(9) . All glycosyltransferases of the Golgi are type II proteins, whose cytoplasmic tail and membrane anchor region are at the NH(2) terminus and whose lumenal portion is divided in a stem region and the COOH-terminal catalytic domain(9) . In recent years some structural requirements for protein localization in the Golgi have been defined. Generally, Golgi localization requires a membrane anchor region flanked by cytoplasmic and luminal protein domains. Specific sequences that determine Golgi localization may be localized in each of these three regions. In mammalian cells, some Golgi proteins contain targeting sequences in the cytoplasmic tail(10) , whereas beta-1,4-galactosyltransferase contains localization sequences in the membrane anchor(11, 12, 13, 14) . beta-Galactoside alpha-2,6-sialyltransferase (ST) and N-acetylglucosaminyltransferase I are the only known enzymes whose localization in the Golgi requires specific luminal sequences(14, 15, 16) . The membrane anchor of ST is needed for localization, but can be replaced by an artificial sequence containing 17 leucine residues (15) or other hydrophobic sequences(17) . The membrane anchor of ST appears to function as a transmembrane spacer, which is required to present targeting sequences on the cytoplasmic and luminal side of ST(17) . Whereas in mammalian cells Golgi targeting sequences may be localized in either cytoplasmic, membrane and luminal sequences, the only defined localization sequences in three Golgi enzymes of yeast (Ste13p, Kex2p, Kex1p) are in the cytoplasmic tails (18, 19, 20) . These results suggest a single Golgi localization mechanism in yeast requiring structural information in the cytoplasmic tails. Cellular components that interact with the membrane anchor regions and effect Golgi localization have not been clearly defined in either mammalian or yeast cells.

It should be emphasized that besides its function in Golgi localization the membrane anchor of type II proteins is also needed as a secretion leader to translocate the respective protein across the membrane of the ER. Some signal sequences of secreted mammalian proteins have been shown to be used and processed in the yeast S. cerevisiae(21) . These findings, as well as the relatively broad range of possible signal sequence structures(22) , suggest that the membrane insertion function of a mammalian membrane anchor region may also be used in yeast. However, it was unclear if the Golgi localization function of the anchor region would be recognized in the heterologous yeast host. In this paper we demonstrate that both the membrane insertion function, as well as the Golgi localization function of a mammalian glycosyltransferase, rat ST, are functional in yeast. This finding suggests that in yeast, specific Golgi-targeting sequences may not be limited to the cytoplasmic tail, as has been reported previously (18, 19, 20) . Thus yeast, as mammalian cells, may utilize several different mechanisms for Golgi localization of proteins.


EXPERIMENTAL PROCEDURES

Strains and Growth Conditions

S. cerevisiae SEY6210 was used as host for recombinant plasmids (MATalpha leu2-3,112 ura3-52 his3-200 trp1-901 lys2-801 suc2-9 GAL) (23) . As a control the mnn9 mutant strain ER112-1B/C3 (MATalpha ura3-52 pep4-3 leu2 mnn9) was used. Strain SEY6210 was transformed with recombinant plasmids as described(24) . Transformants were grown in SD minimal medium or SGal minimal medium (0.67% yeast nitrogen base (Difco), 2% galactose, 0.2% glucose) containing leucine, tryptophane, histidine, and lysine(24) .

Plasmids

Plasmid pbs+St3 containing a 1645-base pair EcoRI fragment encoding rat ST inserted into the EcoRI site of pKS+ (Stratagene) was obtained from J. Paulson (25) . The ST sequence differs from the published sequence (25) by mutation of an A to a G at base number 123, which removes the internal EcoRI sequence without changing the amino acid sequence. Plasmid pRB58 carries the full-length SUC2 gene in YEp24 (revertant of pRB55(26) ). Plasmid pYAAI, which encodes a fusion of the tail and membrane anchor regions of ST, was constructed in several steps. First, a convenient EcoRI restriction site was inserted upstream of the translational start codon of the ST gene by primer-directed mutagenesis. To this effect the 1.6-kilobase EcoRI fragment of plasmid pbs+St3 was inserted into the mutagenesis vector pSELECT, and in vitro mutagenesis was performed using the 21-mer 5`-CAT TAA GAA TTC TCC GAG GAC-3` according to the commercial protocol (Promega). The mutagenesis generates the following sequence (the coding region is in italics).

A 120-base pair EcoRI-DraII gene fragment of the mutated gene encoding the membrane anchor region was then inserted between the EcoRI and SmaI sites of pUC-8 (the DraII site had been filled in using Klenow enzyme). The resulting plasmid, pST-Anker, was cut with BamHI, its 5` overhangs were filled in using Klenow enzyme, and it was then ligated with a 2-kilobase SalI fragment of plasmid pInvSal, which carries a truncated SUC2 gene encoding yeast invertase(27) . In this manner a gene fusion encoding an in-frame fusion between the membrane anchor region of ST and the mature yeast invertase were constructed (plasmid pC). The fusion junction has the following sequence (ST anchor region, bold; SUC2, italics; numbers are residues of mature invertase).

The fusion was excised from plasmid pC as an EcoRI-HindIII fragment and ligated with a 0.5 kilobase BamHI-EcoRI fragment carrying the ACT1 promoter (28) and the large BamHI-HindIII fragment of the centromer vector YCp50(24) . The final vector was designated pYAAI.

Cell Fractionations

Cell fractions were prepared as described (29, 30, 31) with several modifications (method A). Yeast cells were grown in SGal-medium to an A leq 0.8. Spheroplasts were prepared as follows: 100 A units of cells were harvested by centrifugation at 1500 times g for 5 min and preincubated in 100 mM Tris-HCl, pH 9.4, 10 mM dithiothreitol at room temperature for 10 min. Cells were resuspended in TSD buffer (50 mM Tris-HCl, pH 7.5, 1.5 M sorbitol, 10 mM dithiothreitol) and treated with 3000 units of lyticase (Sigma) and 1% glucuronidase (Sigma) for 30-45 min at 30 °C until at least 90% of the cells had formed spheroplasts. Spheroplasts were harvested (2000 times g, 5 min), washed in 3 ml of TSD buffer, and washed again in 20 mM HEPES-KOH, pH 6.8, 150 mM potassium acetate, 5 mM magnesium acetate, 1.5 M sorbitol; spheroplasts were resuspended in 500 µl of this buffer and frozen above liquid nitrogen(32) . To prepare cell fractions the spheroplasts were slowly thawed on ice, harvested (5 min at 1500 times g), and resuspended in 3 ml of TEA buffer (0.8 M sorbitol, 10 mM triethanolamine, 1 mM EDTA, pH 7.6, proteinase inhibitors pepstatin, leupeptin, antipain at 2 µg/ml, 1 mM PMSF). The cell suspension was homogenized by 20 strokes in a precooled tissue homogenizer with a tight-fitting glass pestle and then centrifuged for 3 min at 1000 times g (3000 rpm) in a JA20 rotor (Beckman) to generate crude extract (S1) in the supernatant and a cell pellet (P1). P1 was resuspended in TEA buffer and centrifuged to generate S1`. The combined supernatants were centrifuged for 10 min at 10,000 times g (9000 rpm) in a JA20 rotor to generate supernatant S2 and pellet P2. S2 was then spun for 1 h at 120,000 times g (35,000 rpm) in a Ti50 rotor (Beckman) to generate supernatant S3 and pellet P3. The pellet fractions P2 and P3 containing cellular organelles were resuspended in 1.24 ml of buffer A (55% sucrose, 10 mM HEPES-KOH, pH 7.5, 1 mM EDTA, 1 mM PMSF) and homogenized in the tissue homogenizer with six strokes. In the case of the pYAAI transformant P2 and P3 contained about 45% of the invertase activity of the crude extract. The homogenate was placed at the bottom of a SW41 tube (Beckman polyallomer tube 14 times 95 mm) and overlaid with 1.24 ml portions of buffer A containing 50, 45, 40, 35, 30, or 25% sucrose yielding a discontinuous sucrose gradient. The gradient was centrifuged for 16 h at 170,000 times g in a Beckman SW 40 rotor. After centrifugation 550-µl fractions were collected from the top of the gradient.

In an alternative method (method B) spheroplasts were prepared from 325 A units of transformed cells. Spheroplasts were washed with 1 M sorbitol, resuspended in 0.6 ml of buffer (20 mM Tris-HCl, pH 7.5, 1 mM MnCl(2), 15% sucrose/protease inhibitors leupeptin, pepstatin A, and antipain at 1 µg/ml), and broken using glass beads (, 0.45 mm) at low speed on a Vortex (15-s intervals for a total of 1.5 min, at 4 °C). Cell debris was removed by a low-speed centrifugation (2000 rpm/10 min) and the supernatant containing cytoplasmic proteins and organels was layered on top of a discontinous sucrose gradient (25-50% sucrose) in 13 times 51-mm polyallomer tubes. Following centrifugation in a SW 55 rotor (Beckman) at 42,000 rpm for 15 h, 300-µl fractions were collected from the top of the gradient.

Immunological Procedures

30-50 µl of each sucrose gradient fraction (cell fraction) was separated by SDS-PAGE and proteins were detected by immunoblotting using specific antibodies (33) . Primary antibodies were diluted 1:1250 (anti-invertase) or 1:400 (anti-diaminopeptidyl aminopeptidase A) or 1:500 (anti-Kex2p) or 1:6000 (anti-ATPase); the secondary antibody was goat anti-rabbit coupled to alkaline phosphatase (Bio-Rad), diluted 1:5000. Immunoblots were developed with 5 mg of 5-bromo-4-chloro-3-indolyl phosphate and 70 mg of nitro blue tetrazolium in 50 ml of buffer (0.1 M Tris-HCl, pH 8.8, 0.1 M NaCl, 0.002 M MgCl(2)). Immunoblots were evaluated by densitometry using a Molecular Dynamics laser densitometer. For immunoprecipitations cells were grown in low sulfate medium (5) containing 2% galactose, 0.2% glucose to an A of 0.3-0.6; 5 A units were labeled with 150 µCi of TranS-label (ICN Biomedicals, Inc.) followed by the preparation of cell extracts, essentially as described(5) . Invertase-related proteins were immunoprecipitated by addition of 7.5 µl of anti-invertase antibody followed by the addition of 37.5 µl of protein A-Sepharose (20%), as described(5) . The washed pellet was resuspended in 200 µl of buffer (1% SDS, 0.5% Triton X-100 in phosphate-buffered saline) for 5 min at 95 °C, and to 150 µl of the resulting solution, 600 µl of dilution buffer (5) was added. To 200 µl of the dilution, either 5 µl of anti-invertase antibody or 5 µl of anti-alpha-1,6-mannose were added followed by immunoprecipitation with protein A-Sepharose. The immunoprecipitates were solubilized in sample buffer and separated by SDS-PAGE (7.5% acrylamide, 0.18% bisacrylamide); an equivalent sample of the solubilized first precipitate was added as a control. The resulting SDS-PAGE gels were analyzed by autoradiography.

Assays

NADPH-dependent cytochrome c oxidoreductase(34) , cytochrome c oxidase(34) , and the Golgi marker enzyme Man-Tf (35) were determined essentially as described. alpha-Mannosidase was assayed in 50 mM potassium phosphate, pH 7, 0.04% Triton X-100, 1 mMp-nitrophenyl-alpha-D-mannopyranoside for 2 h at 30 °C and stopped using 2 volumes of 2% Na(2)CO(3)(34) . Invertase activity was determined as described(23) . Protein was determined using the Bio-Rad reagent with bovine serum albumin as standard protein. Digestions with peptide:N-glycosidase F were performed according to the instructions of the manufacturer (New England Biolabs, Inc.). The sucrose concentrations in the gradient fractions were determined using the refractive index.


RESULTS

Construction of a Sialyltransferase-Invertase Fusion

We constructed a gene fusion that encodes the 34 NH(2)-terminal amino acids of rat liver alpha-2,6-ST joined to the mature invertase protein of the yeast S. cerevisiae (Fig. 1). The ST segment contains a presumed 9-amino acid tail, 17 amino acids of the presumed membrane anchor region, which is followed by 8 additional amino acids(25) . The fusion gene was placed under transcriptional control of the yeast ACT1 promoter and inserted into the yeast centromere vector YCp50 that carries the URA3 wild-type gene. The resulting plasmid, pYAAI, was transformed into a yeast ura3 mutant strain (SEY6210) lacking all invertase gene sequences (SUC2^0)(23) , selecting uracil prototrophs. Thus, the only source of invertase in the transformants is the fusion gene on the expression plasmid. Because the invertase signal sequence is missing in the fusion protein, its only possibility to gain entrance to the yeast secretory pathway is the use of ST sequences.


Figure 1: Construction of a yeast expression vector for a ST-invertase fusion. The expression unit consisting of the ACT1 promoter, the sialyltransferase membrane anchor region (ST), and the yeast SUC2 gene was inserted into the centromere vector YCp50 to generate pYAAI. The DNA sequence encoding the ST-invertase fusion junction is shown at the bottom, along with the encoded amino acid residues. The numbers refer to residues of ST or mature invertase, as indicated.



As a control, we used the multicopy yeast vector pRB58, which carries the SUC2 gene encoding the secreted glycosylated form, as well as the cytoplasmic unglycosylated form of invertase(26) . Again, pRB58 was transformed into strain SEY6210 selecting Ura prototrophs.

Glycosylation of the ST-fusion Protein

-In eukaryotic cells N-glycosylation of a protein depends on its entry of the secretory pathway. Furthermore, the extent of glycosyl chain processing indicates how far the respective protein has traversed the secretory pathway. We determined the glycosylation status of the ST-invertase fusion in yeast transformants carrying pYAAI by immunoblotting experiments. In control experiments, transformants carrying plasmid pRB58 (encoding wild-type invertase) were analyzed. Spheroplasts of yeast transformants were prepared and broken by a combination of osmotic lysis and mild mechanical disruption; a crude cell extract was obtained as a supernatant in a low-speed centrifugation step. Furthermore, the periplasmic protein fraction liberated by the lyticase/beta-glucuronidase treatment during spheroplast formation was assayed.

The presence of plasmid pRB58 in transformants leads to the overproduction of the secreted and cytoplasmic forms of invertase(26) . The periplasmic fraction of the pRB58 transformant contained the highly N-glycosylated form of invertase, with a molecular mass of 100-180 kDa (Fig. 2, lane 3), as expected (36) ; in addition, presumably due to cell lysis during spheroplast formation, the cytoplasmic form of invertase was detected, which in our gel system had an apparent molecular mass of about 56 kDa (Fig. 2, lane 3). The periplasmic fraction of the pYAAI transformant did not contain a protein reactive with the anti-invertase antiserum (Fig. 2, lane 2), whereas in the crude extract of this transformant (obtained by method A, see below) a heterogeneous form of invertase with a molecular mass ranging from 90 to 110 kDa, containing at least two distinct invertase species, was found (Fig. 2, lane 5) (heterogeneity of ST-invertase is also shown in Fig. 4, A and B, as discussed below). As expected, a cytoplasmic unglycosylated form of invertase was not detected in crude extracts of the pYAAI transformant, because this is encoded only by the wild-type SUC2 gene(26) .


Figure 2: Invertase production by yeast transformants. 5 µl of the periplasmic fraction (lanes 2 and 3) or 10 µl of a crude extract of yeast transformants (lanes 4-7) were analyzed by immunoblotting using an anti-invertase antibody. lanes 2, 5, and 7, pYAAI transformant; lanes 3, 4, and 6, pRB58 transformant. Samples in lanes 6 and 7 had been treated by peptide:N-glycosidase F. The apparent molecular masses of prestained molecular mass standards (Sigma) are indicated. Symbols on the right indicate the migration of the glycosylated () and deglycosylated (circle) forms of ST-invertase in the pYAAI transformant.




Figure 4: Distribution of anti-invertase-reacting proteins in cell fractions separated by sucrose density gradient centrifugation. Cells were fractionated by method A (A) or by method B (B and C), as indicated in the text. A and B, pYAAI transformant; C, pRB58 transformant. The migration of the core-glycosylated form of wild-type invertase is marked by the asterisk.



By digestions with peptide:N-glycosidase F we examined if the produced invertase forms were N-glycosylated. Treatment of the crude extract of the pRB58 transformant with peptide:N-glycosidase F resulted in the appearance of a distinct invertase species, which had a size about 3 kDa greater than the cytoplasmic form (Fig. 2, lane 6). From the intensities of staining in the immunoblot we estimate that about equal amounts of the cytoplasmic form and the peptide:N-glycosidase F-sensitive N-glycosylated form are present in the pRB58 transformant (in which invertase is overproduced compared with a wild-type Suc2 strain). The fact that deglycosylated invertase has a greater size than cytoplasmic invertase may be due to O-glycosylation of secreted invertase (37) or the presence of a short N-glycosyl chain remaining after peptide:N-glycosidase F digestion. The ST-invertase fusion in the pYAAI transformant appeared quantitatively N-glycosylated, because peptide:N-glycosidase F treatment resulted in the disappearance of the 90-110-kDa form (Fig. 2, lane 5) and the appearance of two distinct invertase species of about 60 and 63 kDa (Fig. 2, lane 7). The presence of 34 amino acids of ST at the NH(2) terminus of invertase is expected to increase its size by about 3 kDa; thus, the 60-kDa form of the ST-fusion, which comigrates with deglycosylated wild-type invertase, appears to represent the unmodified fusion protein. The 3-kDa increase in molecular mass of the 63-kDa form may be caused by O-glycosylation, as discussed for wild-type invertase. Partial O-glycosylation of proteins secreted by yeast has been reported previously(38, 39) .

To explore how far ST-invertase had traversed the secretory pathway, we performed immunoprecipitations to determine if ST-invertase had received alpha-1,6-mannose linkages, a modification which occurs in the Golgi compartment(4) . Cells were labeled with [S]methionine, and invertase species were immunoprecipitated using anti-invertase antibody. Equal amounts of the resolubilized precipitate were immunoprecipitated with anti-invertase- or anti-alpha-1,6-mannose antibody, respectively, and analyzed by SDS-PAGE followed by autoradiography. As in the above immunoblots (Fig. 2) ST-invertase could be detected using anti-invertase antibody as a smear of proteins containing two distinct proteins (Fig. 3, lanes 1 and 2). Using anti-alpha-1,6-mannose antibody only the predominant upper band and the smear of proteins with higher molecular masses could be precipitated (Fig. 3, lane 3). By scanning of the autoradiographic film we estimate that about 70% of the ST-invertase has obtained Golgi-specific alpha-1,6-mannose modification of its glycosyl chains. An analogous experiment was carried out for the pRB58 transformant, in which the anti-alpha-1,6-antibody was shown to detect only the heterogeneous N-glycosylated form, as expected (Fig. 3, lanes 4-6).


Figure 3: Immunoprecipitation of invertase in yeast transformants. Transformants carrying pYAAI or pRB58 were labeled with [S]methionine, and invertase was immunoprecipitated using an anti-invertase antibody. The precipititate was solubilized, and aliquots were immunoprecipitated with either anti-invertase antibody or anti-alpha-1,6-mannose antibody. Immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. The size of molecular mass standards is indicated. Lanes 1-3, pYAAI transformant; lanes 4-6, pRB58 transformant; lanes 1 and 4, first immunoprecipitate; lanes 2 and 5, consecutive immunoprecipitate with anti-invertase antibody; lanes 3 and 6, consecutive immunoprecipitate with anti-alpha-1,6-mannose antibody. The migration of ST-invertase (), and the cytoplasmic (circle) and glycosylated forms (up triangle) of invertase are indicated.



These results demonstrate that the ST-membrane anchor region is able to direct the invertase reporter protein into the yeast secretory apparatus, where it gets quantitatively N- and, possibly, partially O-glycosylated. The size of >90 kDa of the ST-invertase fusion protein and its heterogeneity are greater than expected from the modification by only core glycosyl chains, which are characteristic of the endoplasmic reticulum. Core-glycosylated invertase has a size of about 90 kDa(36) ; experimentally, we could show that invertase in a mnn9 mutant (4) is smaller and less heterogeneous than the ST-invertase fusion protein (data not shown). Furthermore, we show that at least about 70% of the total ST-invertase molecules have reached a Golgi compartment, since they contain alpha-1,6-mannose determinants.

Localization of the Fusion Protein in the Golgi

The experiments described above suggested that the major portion of the ST-invertase fusion had reached a Golgi compartment, although a precise assignment of its final intracellular localization could not be made. The localization of the minor ST-invertase portion, which did not receive alpha-1,6-mannose modifications, also was not known. Therefore, we performed cell fractionation experiments using sucrose density gradients to demonstrate cofractionation of the ST-invertase fusion with the ER, the vacuole, the plasma membrane, or Golgi compartments, which all could conceivably host the ST-invertase fusion. During the course of these experiments we found that two types of procedures for cell fractionation were necessary to obtain clear evidence of the localization of ST-invertase in the Golgi.

In the first procedure (method A) spheroplasts of the pYAAI transformant were broken by a combination of osmotic lysis and mild mechanical disruption using a tissue homogenizer(31) . The organelle fraction of the cells was then fractionated on a sucrose gradient, and marker proteins in the fractions were assayed immunologically or by their enzymatic activity. An immunoblot on the distribution of ST-invertase in the gradient fraction is shown in Fig. 4A and represented graphically in Fig. 5C. ST-invertase fractionated at intermediate as well as high sucrose density in the gradient, clearly different than the ER marker NADPH-dependent cytochrome c oxidoreductase that migrated in two peaks of different densities (Fig. 5B). Thus, the ST-invertase fusion did not simply obtain Golgi extension of its glycosyl chains and was retrieved to the ER(40) . However, the distribution of ST-invertase at intermediate density had the same relatively broad peak profile as the Golgi marker enzyme Man-Tf, whereas the peak of the late Golgi marker Kex2p appeared more narrow (Fig. 5C). It has been reported previously that subcellular fractionation can separate a Kex2p-containing late Golgi compartment from an intermediate, Man-Tf- and GDPase-containing compartment(7, 29) . Thus, since the vacuolar marker alpha-mannosidase fractionates at the bottom of the gradient (Fig. 5B), it appears that the portion of ST-invertase migrating at intermediate density is localized in the Golgi, possibly in a medial Golgi compartment. Differences between the distribution of ST-invertase and Man-Tf in the gradient occur at higher densities, where Man-Tf has a minor peak (fraction 14) that does not occur with ST-invertase (Fig. 5C); more significantly, a peak at the bottom of the gradient is detected only for ST-invertase and, because of its colocalization with the alpha-mannosidase peak, indicates the presence of the fusion protein in the vacuole. If these assignments are correct, we can estimate from the data in Fig. 4A that about 70% of the ST-invertase fusion produced by the pYAAI transformant are localized in Golgi vesicles of intermediate density, and about 30% are localized in the vacuole. The vacuole may be the ``default'' compartment for ST-invertase, as for yeast Golgi proteins(19, 20, 41) . Furthermore, since the distinct bands visible in the immunoblot of the crude extract (Fig. 2) are distributed with equal intensities across the gradient, it appears that even the portion of ST-invertase that did not receive alpha-1,6-mannose antigenic determinants (lower band, see above) is localized either in a Golgi or vacuolar compartment.


Figure 5: Invertase and marker proteins in cell fractions of a pYAAI transformant obtained by sucrose gradient centrifugation. Organelles of a pYAAI transformant prepared by method A (see text) were separated on a sucrose gradient, and gradient fractions were analyzed for the presence of invertase and marker proteins by immunoblotting or by enzymatic activity, as indicated. Oxidoreductase, NADPH-dependent cytochrome c oxidoreductase; mannosidase, alpha-mannosidase.



To confirm the results of the cell fractionation shown in Fig. 4A, we used an alternative procedure, in which spheroplasts were broken by a brief agitation with glass beads (method B). In this case, the crude extract, including the cytoplasmic fraction, was separated on a sucrose density gradient. Immunoblots on the distribution of invertase in gradient fractions of a pYAAI transformant and a pRB58 transformant are shown in Fig. 4, B and C, respectively; a graphic representation of the pYAAI transformant data is shown in Fig. 6. Using this method it was possible to clearly separate the plasma membrane (marker enzyme ATPase) from other cell organelles (Fig. 6C), which did not succeed satisfactorily with method A. ST-invertase fractionated at intermediate density (around fraction 8), indicating that the fusion is not located in the plasma membrane migrating at high density (Fig. 6C) or in the ER, which fractionated in two peaks of different densities in the gradient (Fig. 6B). The first ER-peak (around fraction 5) comigrated with the cytoplasmic and core-glycosylated, ER forms of invertase in a separate gradient on a pRB58 transformant (Fig. 4C, ER-form marked by an asterisk). As expected, ST-invertase also did not cofractionate with the mitochondrial marker enzyme cytochrome c-oxidase, of which highest activities were found in fractions 11 and 12 (data not shown). On the other hand ST-invertase was distributed very similar to the Golgi marker DPAP A in the gradient (Fig. 6C), although a separation of DPAP A from the vacuolar marker alpha-mannosidase was not possible (Fig. 6B); most likely in method B the vacuole had been sheared into smaller vesicles by the glass bead breakage of the spheroplasts. Thus, from the data of Fig. 6alone it cannot be decided if ST-invertase is localized in the Golgi or the vacuole. However, in combination with the results of the first gradient (Fig. 5), these data clearly demonstrate that the ST-invertase fusion is localized mainly in the Golgi and to a lesser extent in the vacuole. The cytoplasma, the ER, the plasma membrane, and the mitochondria do not contain the ST-invertase fusion.


Figure 6: Invertase and marker proteins in cell fractions of a pYAAI transformant obtained by sucrose gradient centrifugation. Crude extracts prepared by method B (see text) were separated on a sucrose density gradient, and gradient fractions were analyzed for the presence of invertase and marker proteins by immunoblotting or by enzymatic activity (expressed in milliunits/fraction), as indicated. Oxidoreductase, NADPH-dependent cytochrome c oxidoreductase; mannosidase, alpha-mannosidase; ATPase, plasma membrane ATPase; DPAP A, dipeptidyl aminopeptidase A.




DISCUSSION

alpha-2,6-ST has been mainly localized in the trans-Golgi and trans-Golgi network compartments of mammalian cells; in addition, some mammalian cells produce ST in other Golgi compartments or as a secretory form lacking the NH(2)-terminal 63 amino acids(9) . In the present study we constructed a gene fusion encoding a protein, in which the NH(2)-terminal 34 amino acids of rat ST are joined to the yeast protein invertase lacking a secretion leader (26) . The NH(2)-terminal 34 residues of ST contain 9 amino acids of a presumed cytoplasmic tail, 17 amino acids of a membrane anchor region, and a small fragment of the stem region consisting of 8 amino acids(25) . In yeast transformants the fusion protein gets extensively N-glycosylated, indicating that the fusion protein has entered the secretory pathway and is oriented to the lumen of secretory compartments. The lengths of the N-glycosyl chains of the fusion are intermediate between the ER form, which is similar to the form produced by the yeast mnn9 mutant (4) and the fully extended form that occurs on wild-type secreted invertase. This finding indicates that the fusion has reached a compartment subsequent to the ER; the detection of alpha-1,6-mannose linkages in N-glycosyl chains of about 70% of ST-invertase indicates that it has reached the Golgi. Conceivably, besides Golgi compartments the final localization of ST-invertase can be the vacuole, which is the main default compartment for yeast Golgi proteins that are not properly retained (19, 20, 41) or the plasma membrane(10) . Also, it had to be considered that some proteins, after having reached the Golgi and obtained specific Golgi modification, may return to the ER (40) . Therefore, we performed cell fractionation experiments using sucrose density gradients to assign the ST-invertase fusion to a specific intracellular organelle. Using two different procedures for cell fractionation, we obtained clear evidence the major portion of the fusion, about 70%, is located in the Golgi, whereas the remaining portion is associated with the vacuole. The fractionation of ST-invertase closely resembled alpha-1,2-mannosyltransferase, which is located in an intermediate compartment of the yeast Golgi, but was slightly different from the fractionation of Kex2p that is situated in a late Golgi compartment(6) . Therefore, we conclude that the ST membrane anchor region has targeted the invertase reporter protein to the yeast Golgi, possibly the medial Golgi compartment. As stated above, this finding agrees with previous results that the localization of ST in mammalian cells is cell type-specific and may include compartments other than the trans-Golgi or the trans-Golgi network(9) .

The fact that the membrane anchor region of rat ST is able to direct a protein to the yeast secretory pathway is not unexpected, since many different hydrophobic sequences are recognized as signal sequences (22) ; in addition, mammalian signal sequences have been reported to function in yeast(21) . However, efficient Golgi-targeting by this region is surprising, since in yeast Golgi-targeting sequences have been defined for the cytoplasmic tail of Golgi proteins(18, 19, 20) . The cytoplasmic tail of ST is short, comprising only 9 amino acids, and does not contain the consensus sequence for yeast Golgi localization, Y/F-X-Y/F(20) . In mammalian cells, the cytoplasmic tail of ST can be replaced by unrelated sequences, while retaining Golgi localization(15) . Likewise, the membrane anchor region can be replaced by an artificial hydrophobic sequence containing 17 leucine residues or by other hydrophobic sequences(17, 19) . However, all ST-derived fusions that are efficiently targeted to the Golgi retain various lengths of the luminal stem region(14, 19) . In a recent report it has been shown that the 5 amino acids KKGSD of the ST luminal region are sufficient for the targeting function; the two lysines within this sequence appear critical(17) . According to this study the functional role of the membrane anchor may be to act as a transmembrane spacer that correctly positions targeting sequences containing lysine residues in the cytoplasmic and luminal domains(17) . We report here that the cytoplasmic domain, the membrane anchor, and only 8 amino acids of the luminal domain of rat ST, which includes the KKGSD sequence at positions 27-31, are sufficient to direct the invertase reporter protein to the Golgi of the heterologous yeast host. This result suggests that the Golgi-targeting mechanisms for ST in mammalian cells and for the ST fusion protein in yeast are similar. Interspecies function of a Golgi targeting signal is in agreement with a recent model for Golgi targeting, which relies on a sterol gradient along the secretory pathway(42) . Thus, the relatively short ST membrane anchor would not be able to partition into membranes of yeast secretory vesicles destined for the plasma membrane because of their high sterol (ergosterol) levels; in consequence ST would remain in Golgi membranes that are relatively thinner due to their low ergosterol content. Oligomerization has been discussed as another mechanism contributing to Golgi retention(17, 43) . Possibly, the ability of invertase to form homo-oligomers (44) also contributed to the observed Golgi retention, although the formation of hetero-oligomers with resident Golgi proteins cannot be excluded. In separate experiments we found that full-length ST was able to enter the yeast secretory pathway, but was not transported beyond the endoplasmatic reticulum, (^2)a finding which was also reported recently for human alpha-2,6-ST(45) . These results demonstrate that the reporter protein may also have a negative effect on intracellular targeting. Finally, a Golgi retention machinery consisting of specific retention proteins that are conserved between yeast and mammalian cells is consistent with our results.

Thus, yeast cells as mammalian cells may have several mechanisms to localize membrane enzymes to the Golgi compartment. Besides specific sequences in the cytoplasmic tail of proteins(20) , such sequences may also reside in the membrane anchor and the luminal domain. An example for a yeast protein targeted by such a mechanism may be the alpha-1,2-mannosyltransferase (Mnt1p) required for extension of O-glycosyl chains in the yeast Golgi, which is a type II protein with a short cytoplasmic tail of 11 amino acids (35) lacking the consensus sequence Y/F-X-Y/F. Although specific sequences in the Mnt1p membrane anchor region required for Golgi localization have not been defined, it has been shown recently that the transmembrane region is required(46) . Both types of Golgi retention mechanisms may be distinguished by the consequences of overexpression of respective proteins: ST does not bypass the Golgi when overexpressed in mammalian cells, but appears to backup along Golgi compartments and into the ER(15, 47) . On the other hand, overexpression of Kex2p in yeast and TGN38 in mammalian cells leads to localization to the vacuole or, respectively, the plasma membrane(10, 19) . In our experiments ST-invertase was localized in part in the vacuole, confirming the vacuole as the default compartment for yeast Golgi proteins. The reason for the partial vacuolar targeting of the ST-invertase fusion may be its overexpression beyond the retention capacity of the Golgi (46) ; alternatively, the mammalian Golgi-targeting signal may not be properly recognized in yeast. Possibly, the concept of the yeast Golgi may become more refined with the help of mammalian Golgi proteins. Yeast genetics can then be used to investigate cellular events related to the different Golgi localization mechanisms.


FOOTNOTES

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

§
To whom correspondence should be addressed: Institut für Mikrobiologie, Heinrich-Heine-Universität, Universitätsstr. 1/26.12, D-40225 Düsseldorf, Germany. Tel.: 49-211-311-5176; Fax: 49-211-311-5176; Joachim.Ernst{at}uni-duesseldorf.de.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; GDPase, guanosine diphosphatase; Man-Tf, alpha-1,2-mannosyltransferase; PMSF, phenylmethylsulfonyl fluoride; ST, beta-galactoside alpha-2,6-sialyltransferase (EC 2.4.99.1).

(^2)
C. Lorenz and J. F. Ernst, unpublished results.


ACKNOWLEDGEMENTS

We thank J. Paulson (La Jolla, CA) for the clone containing the sialyltransferase gene. Antibodies against Kex2p, DPAP A, ATPase, and alpha-1,6-mannose were kindly supplied by D. Gallwitz, T. Stevens, A. Goffeau, and A. Franzusoff, respectively. We thank M. Gerads for excellent technical assistance.


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