(Received for publication, July 7, 1994; and in revised form, November 28, 1994)
From the
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
-galactoside
-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
-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.
The Golgi comprises cellular compartments that secreted proteins
traverse between the endoplasmic reticulum (ER) ()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
-factor pheromone and killer factor occur in the
yeast Golgi(5) . The yeast Golgi is subdivided in an early
compartment containing
-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 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
-1,4-galactosyltransferase contains
localization sequences in the membrane
anchor(11, 12, 13, 14) .
-Galactoside
-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.
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.
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
, 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
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.
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.
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 (
) 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
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
-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-
-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-
-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
-1,6-mannose modification of its glycosyl chains.
An analogous experiment was carried out for the pRB58 transformant, in
which the anti-
-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-
-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-
-1,6-mannose
antibody. The migration of ST-invertase (
), and the cytoplasmic
(
) and glycosylated forms (
) 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 -1,6-mannose
determinants.
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 -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
-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
-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,
-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 -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, -mannosidase; ATPase, plasma
membrane ATPase; DPAP A, dipeptidyl aminopeptidase
A.
-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
-terminal 63 amino acids(9) . In the present
study we constructed a gene fusion encoding a protein, in which the
NH
-terminal 34 amino acids of rat ST are joined to the
yeast protein invertase lacking a secretion leader (26) . The
NH
-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
-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
-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, ()a finding which was also reported recently for
human
-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 -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.