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INTRODUCTION |
Glycosylation of proteins and lipids occurs in the endoplasmic
reticulum and the Golgi apparatus and is mediated by
glycosyltransferases and glycosidases that are resident there. The
enzymes are ordered so that carbohydrates can be sequentially added to
and removed from proteins and lipids (1-3). The mechanism for
retention of the enzymes in the appropriate subcompartment and the
domains of the enzymes that direct this localization are not clear. At present there are two proposed mechanisms for Golgi glycosyltransferase localization, kin recognition (4, 5), where like gycosyltransferases form aggregates within the Golgi, and the lipid bilayer model, where
the hydrophobic transmembrane domain retains the transferases in the
Golgi (6). Neither the kin-recognition nor the lipid bilayer models
fully explain the specific and functional localization of transferases
to discrete sites within the Golgi; we now demonstrate that the
cytoplasmic tail is a key element in the specific localization of
1,2-fucosyltransferase.
Glycosyltransferases are type II integral membrane proteins, with the
catalytic domain residing in the lumen of the Golgi (7) and the N
terminus in the cytoplasm (cytoplasmic tail). Several
glycosyltransferases have been localized in compartments of the Golgi
by electron microscopy (for review see Ref. 8). Studies of
glycosyltransferases have demonstrated the importance of the
transmembrane domain and its flanking sequences (9-14) and the luminal
domain (15) for localization.
We examined two glycosyltransferases that compete for the same
acceptor, N-acetyllactosamine
(NAcLac),1 and showed that
when there is expression of
1,3-galactosyltransferase (GT) together
with
1,2-fucosyltransferase (FT) within a cell, the FT predominates,
to produce H substance rather than Gal
(1,3)Gal on the cell
surface (16). Furthermore, switching the cytoplasmic tails of these
transferases altered the dominant effect of FT indicating that the
cytoplasmic tail affects the localization sites of the enzymes
(17).
Here we examine the role of the cytoplasmic tail of FT for functional
localization in the Golgi using the model system of competition with GT
for acceptor and show in functional assays and by microscopy that the
cytoplasmic tail is essential for FT localization as the ability to
compete with GT was abolished and the localization of the enzyme
changed. In addition, the amino acids in the tail conferring
localization were defined by mutagenesis.
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EXPERIMENTAL PROCEDURES |
DNA Constructs--
Untagged GT (18) and FT (19) were as
described. The cytoplasmic tail FT mutants used the following
oligonucleotides as the 5' PCR primer (the mutated amino acid is
lowercase): FTtrp2ala, 5'-GCGGATCCATGgctGTCCCCAGCCGCCGCCACCTCTGTCTG;
FTval3ala, 5'-GCGGATCCATGTGGgctCCCAGCCGCCGCCACCTCTGTCTG; FTpro4ala,
5'-GCGGATCCATGTGGGTCgctAGCCGCCGCCACCTCTGTCTG; FTser5ala, 5'-GCGGATCCATGTGGGTCCCCgctCGCCGCCACCTCTGTCTG; FTarg6ala,
5'-GCGGATCCATGTGGGTCCCCAGCgctCGCCACCTCTGTCTG; FTarg7ala,
5'-GCGGATCCATGTGGGTCCCCAGCCGCgctCACCTCTGTCTG; FThis8ala, 5'-GCGGATCCATGTGGGTCCCCAGCCGCCGCgctCTCTGTCTG; FTser5thr,
5'-GCGGATCCATGTGGGTCCCCactCGCCGCCACCTCTGTCTG; and FTser5tyr,
5'-GCGGATCCATGTGGGTCCCCtacCGCCGCCACCTCTGTCTG. The 3' primer was
5'-GCTCTAGAGGTTCAAGGCCCAGCCAACATCTGGAGTGGAGACAAGTC, and the FT template
was as described (19).
For the FLAG-tagged mutants the PCR 5' primers were as follows:
FT-FLAG, 5'-GCGGATCCATGTGGGTCCCCAGCCGCCGCCACCTC; FT
cyt,
5'-GCGGATCCATGCACCTCTGTCTGACCTTCCTGCTAGTCTGTG; and the 3'primer
incorporating the FLAG epitope was
5'-CGTCTAGATCACTTGTCATCGTCGTCCTTGTAGTCAGGCCCAGCCAACATCTGGAGTGGAGAC-3'. After the initiation methionine, FT
cyt starts at His8
of the FT amino acid sequence.
The ftGT chimera was produced by polymerase chain reaction
using the untagged pig
1,3-galactosyltransferase (18) as
template. The oligonucleotides were
5'-GCGGATCCATGTGGGTCCCCAGCCGCCGCCACGTGGTTCTGTCAATGCTGCTTGTC-3' and
5'-GCTCTAGAGCGTCAGATGTTATTTCTAACCAAATTATAC-3'. All DNA was digested
with BamHI and XbaI and ligated into pcDNA1.
Cell Culture and Transfection--
DNA was purified using Qiagen
products and transfected into COS-7 cells using DEAE-dextran (20) or
LipofectAMINE Plus (Life Technologies, Inc.) as recommended by the
manufacturer. COS-7 cells were grown in Dulbecco's modified Eagle's
medium (CSL) supplemented with 10% fetal calf serum (CSL).
Immunofluorescence and Confocal Microscopy--
COS-7 and
Chinese hamster ovary cells transformed with large T antigen (21) were
stained 48 h after transfection by incubation in antibody or
lectin-diluted in phosphate-buffered saline/0.5% bovine serum albumin.
For immunofluorescence microscopy counting was performed blind, and
experiments were repeated at least three times; flow cytometry was
performed at least three times busing a Becton Dickinson FACSCalibur.
For confocal microscopy, cells were passaged into chamber slides and
cultured overnight prior to incubation for 4 h in cycloheximide
(100 µg/ml) to inhibit protein synthesis, followed by a 15-min
incubation in Brefeldin A (5 µg/ml). Cells were fixed in 2%
paraformaldehyde/phosphate-buffered saline followed by permeabilization
and incubation in 0.5% saponin/phosphate-buffered saline containing
diluted antibodies or lectins. The FLAG epitope was detected with
anti-FLAG monoclonal antibody M2 (Sigma) followed by sheep anti-mouse
Ig conjugated to FITC (Silenus). The cells were mounted in Prolong
(Molecular Probes). Griffonia (Bandeiraea) simplicifolia lectin 1 (IB4) and Ulex europaeus
agglutinin 1 (UEA1) (Sigma) were conjugated to FITC and used to stain
for the products of GT, Gal
(1,3)Gal, and FT (H substance), respectively.
Enzyme Assays--
The assays for
1,2-fucosyltransferase and
1,3-galactosyltransferase were as described (16).
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RESULTS |
Removal of the FT Cytoplasmic Tail Reduces the Ability of the
Enzyme to Compete for Acceptor--
The strategy used was to alter FT
and perform the following two studies: (a) measure the
function of FT by its effect on Gal
(1,3)Gal expression on the cell
surface and (b) examine cells by confocal microscopy to
localize the enzymes. When GT (which attaches Gal
(1,3)Gal linkages to NAcLac) and FT (which attaches
1,2-fucose to NAcLac creating H substance) are coexpressed, FT fucosylates NAcLac, in
preference to GT, and this reduces the level of Gal
(1,3)Gal (the
product of GT) on the cell surface (16).
To establish the functional assays, DNA constructs (Fig.
1) were transfected into Chinese hamster
ovary cells transformed with large T antigen that were analyzed by flow
cytometry (Fig. 2, A-J) for
Gal
(1,3)Gal staining (the enzymatic product of GT), using IB4-FITC
(Fig. 2, A, C, E, G, and
I) or for H substance (the enzymatic product of FT), using
UEA1-FITC (Fig. 2, B, D, F,
H, and J).

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Fig. 2.
Determination of the ability of
FT cyt to compete with GT for acceptor.
Chinese hamster ovary cells transformed with large T antigen were
cotransfected with DNA encoding the following: A and
B, vector + GT; C and D, vector + FT;
E and F, vector + FT cyt; G and
H, GT + FT; and I and J, GT + FT cyt. The cells were analyzed by flow cytometry after surface
staining for (i) the products of GT and Gal (1,3)Gal detected with
IB4-FITC and (ii) the products of FT and H substance detected with
UEA-1-FITC. The marker set for the population of positively stained
cells is shown with the mfu.
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Several important findings were apparent from these cotransfection
experiments. Cells transfected with GT led to expression of
Gal
(1,3)Gal where the positive cells (shown by the marker on each
panel) had 250 median fluorescence units (mfu; see Fig. 2A);
these cells were nonreactive with UEA-1 lectin detecting H substance
(Fig. 2B). Cells transfected with FT gave reciprocal results, negative for Gal
(1,3)Gal (with IB4; see Fig. 2C)
and positive for H substance (281 mfu with UEA1; see Fig.
2D). When the two transferases were present simultaneously,
the level of Gal
(1,3)Gal was markedly reduced (63 mfu; see Fig.
2G) compared with GT expressed alone (250 mfu; see Fig.
2A); there was no decrease observed in the level of H
substance (297 mfu; see Fig. 2H versus 281 mfu;
see Fig. 2D). These findings were previously interpreted as
being due to the localization of FT in an earlier compartment of the
Golgi than GT (16, 17). Thus the system was in place to examine
alterations in the localization of the FT after removing the
cytoplasmic tail.
When the cytoplasmic tail was removed from FT (FT
cyt) there was
reduced but not absent expression of H (158 mfu; see Fig. 2F) compared with full-length FT (281 mfu; see Fig.
2D) and no expression of Gal
(1,3)Gal (Fig.
2E); this was not because of different levels of the
transferases expressed, which were similar as shown by Western blot
(data not shown). However, there was appreciable surface staining for H
substance indicating that FT
cyt was functional. Clearly the lack of
the FT cytoplasmic tail was affecting FT function and perhaps its
localization. This was confirmed in coexpression studies with GT, as
FT
cyt was no longer able to decrease Gal
(1,3)Gal expression (173 mfu; see Fig. 2I) to the same extent as full-length FT (63 mfu; see Fig. 2G). Therefore FT
cyt is present (as H is
present), but as it no longer affects Gal
(1,3)Gal expression, it is
unlikely to be acting on the acceptor before GT, suggesting it has
moved elsewhere. Thus the two enzymes, FT and GT, no longer have the
same relationship, and FT
cyt is no longer acting on the acceptor
prior to GT. Indeed, GT appears to act before FT
cyt, as the amount
of H substance on the surface of the cells when GT and FT
cyt were
coexpressed (92 mfu; see Fig. 2J) was reduced compared with
FT
cyt alone (158 mfu; see Fig. 2F) showing that GT is now
acting on the acceptor before FT.
What has happened to the FT after removal of the tail? It is certainly
present in the Golgi (see also below) as it is able to fucosylate
proteins (UEA1-positive staining). We suspect it has moved to a
post-Golgi compartment as the level of UEA1 staining is less than when
the tail is present and GT, which is in the trans Golgi, is able to act
on the acceptor before the FT
cyt. It was therefore important to
directly examine the localization of FT after removal of the tail
(FT
cyt).
The Intracellular Distribution of FT Is Changed after the
Cytoplasmic Tail Is Removed--
Fluorescent microscopy was used on
cycloheximide-treated cells, which were subsequently stained for the
transferases. The appearance of the Golgi and TGN can be distinguished
using Brefeldin A treatment (22). When cells are stained for Golgi
prior to Brefeldin A treatment, they have a juxtanuclear staining
pattern, which is lost after Brefeldin A treatment, to become diffuse
cytoplasmic staining indicative of endoplasmic reticulum (22). However, the TGN, which also has a juxtanuclear staining pattern without Brefeldin A, collapses around the microtubule organizing center with
Brefeldin A and appears as a bright area of staining, similar to that
observed for the microtubule organizing center (22). When
FT-FLAG was examined, the juxtanuclear staining pattern
(suggesting either Golgi or TGN localization) was observed without
Brefeldin A treatment (Fig.
3A) and was altered to a
diffuse cytoplasmic staining pattern after Brefeldin A treatment (Fig.
3B), showing that FT was present in the Golgi, a finding in
agreement with others (15). In contrast, when the FT tail was removed,
FT
cyt staining appeared as juxtanuclear (Fig. 3C),
suggesting either Golgi or TGN localization, but after Brefeldin A
treatment the staining pattern was similar to that of the TGN (Fig.
3D). It is likely that FT
cyt is in the TGN, because
Brefeldin A causes the TGN to collapse around the microtubule
organizing center (22), and in our study this is the appearance
observed in the cells. No cell surface staining for the FLAG tag was
observed in either FT- or FT
cyt-transfected cells (data not shown).
Thus after removing the FT tail, FT has moved from the Golgi to the
TGN. The microscopic and functional studies show that the
cytoplasmic tail is responsible for the specific localization of FT in
the Golgi, and after its removal, the enzyme is found in the TGN.

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Fig. 3.
Localization of FT and
FT cyt in transfected and cycloheximide ± Brefeldin A-treated COS-7 cells. Permeabilized cells were
stained for the FLAG epitope. FT is localized to the Golgi in
cylcoheximide-treated cells (A), and after treatment with
Brefeldin A (B) FT is localized to the endoplasmic
reticulum. FT cyt is localized to the Golgi in cylcoheximide-treated
cells (C), and after treatment with Brefeldin A
(D) some of the FT is localized to the endoplasmic reticulum
with an additional small region of intense staining consistent with TGN
staining.
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Ser5 of the FT Cytoplasmic Tail Is Essential for
Functional Golgi Localization--
Having shown the importance of the
FT cytoplasmic tail, we sought to determine which of the 8 amino acids
(MWVPSRRH) in the cytoplasmic tail sequence of FT are important for its
localization. Using the same functional readout each amino acid of the
cytoplasmic tail was mutated and was examined for function. Thus, it
would be expected that mutation of an amino acid in the FT tail, which is important for localization, would result in a reduced ability of FT
to compete for acceptor with GT and result in a subsequent increase in
the level of Gal
(1,3)Gal. Each amino acid of the cytoplasmic tail
was substituted by Ala to produce constructs FTtrp2ala, FTval3ala,
FTpro4ala, FTser5ala, FTarg6ala, FTarg7ala, and FThis8ala. These
constructs, full-length FT, or vector were cotransfected with GT into
COS-7 cells, and the number of cells showing surface staining for
IB4-FITC or for UEA-1-FITC was counted (Fig.
4A). In cells transfected with
FT or each of the FT mutants, 60% of cells were stained with UEA-1,
showing that FT was present and functional. However, differences in the
ability of the mutants to inhibit GT activity (as measured by cell
surface IB4 staining) were found. Using GT alone, 50% of cells were
positive for Gal
(1,3)Gal, and this was reduced to 20% by
coexpression of FT. The FTval3ala and FThis8ala mutants also inhibited
Gal
(1,3)Gal expression to 20%, i.e. these
mutations had no effect on FT function and therefore localization and
were unlikely to be involved in the localization of the enzyme.
However, transfection of the FTtrp2ala, FTpro4ala, FTarg6ala, and
FTarg7ala mutants with GT resulted in 40% of cells expressing
Gal
(1,3)Gal, indicating some alteration of FT localization. However,
the Ser5 to Ala mutation removed the inhibitory effect of
FT on GT as staining for Gal
(1,3)Gal was on 50% of cells, and
therefore there was no competition between the enzymes. Thus of the 8 amino acids, positions 4-7 are involved in localization of the enzyme.
Ser5 is the most important amino acid; for position 3, a
Val to Ala substitution is not a major structural change and could be
involved in FT localization.

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Fig. 4.
Determination of the ability of FT mutants to
compete with GT or ftGT for acceptor in COS-7 cells. COS-7 cells
were cotransfected with wild-type or mutant FT and (A) GT or
(B) ftGT. The percentage of cells expressing (i) the
products of GT and Gal 1,3Gal and detected with IB4-FITC and (ii) the
products of FT and H substance and detected with UEA1-FITC are shown.
DNA transfection combinations are shown.
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To confirm that these differences were not because of changes in the
enzymatic activity caused by mutation to the cytoplasmic tail, enzyme
assays were performed. We have previously shown that changes in the
levels of Gal
1,3Gal and H substance on the cell surface were not
because of changes in enzymatic activity (16) and that chimeric
glycosyltransferases did not exhibit altered kinetics of enzyme
function after switching of cytoplasmic tails between GT and FT (17).
However the issue of expression level of the FT mutants in the
current experiments needed to be addressed. Cells were transfected as
described for Fig. 4A, and lysates were assayed
(Table I). There were no
significant differences in the enzymatic rates between any of the FTs
or the rate of GT (this was expected as the same amount of GT encoding
DNA was transfected into each dish of cells). In particular, the S5A
mutant had an enzyme rate of 1216.4 ± 206.7 pmol/hr/mg compared
with that of FT, which was 1212.7 ± 206.1 pmol/hr/mg. Thus, the
increase in Gal
1,3Gal is not because of a decrease in the level of
enzyme activity of the FT mutant.
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Table I
Enzymatic rates of 1,2 fucosyltransferase in COS cell lysates
Cells were cotransfected with GT and wild-type or mutant FTs. Enzymatic
rate is the mean ± S.D. of two independent experiments. The mean
enzymatic rate for GT was 5.972 ± 1.166 pmol/hr/mg.
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The mutations in the cytoplasmic tail could lead to subtle shifts in FT
localization. However, the assay described above (Fig. 4A)
could not detect subtle differences as the number of cells expressing H
substance was similar for all the mutants (Fig. 4A). To
investigate possible subtle changes a new system was developed that is
vulnerable to minor alterations in FT function. A construct was
produced, ftGT, which encodes the 8 amino acid cytoplasmic tail of FT,
but not the transmembrane and luminal domains of GT. Based on
earlier work using the human FT cytoplasmic tail (17), ftGT would be
positioned in the same subcompartment as FT and would compete for
acceptor equally as both transferases have the same cytoplasmic tail.
Using coexpression of ftGT and the FT mutants, any physical shift of FT
mutants from the functional site in the Golgi would reduce their
ability to compete for acceptor with ftGT and would therefore result in
a loss of UEA-1 staining. ftGT was cotransfected with the Ala mutant
FTs, wild-type FT, or vector alone, and cell surface staining for IB4
and UEA-1 was determined (Fig. 4B). FT did not inhibit the
level of Gal
(1,3)Gal after cotransfection with ftGT (60% of cells
were positive), but rather both enzymes were able to function equally
as they are probably located in the same subcompartment. Similarly,
none of the mutants altered the level of cell surface H substance (60%
of cells) except for FTser5ala, which reduced the level of cell surface
staining from 60 to 30%. This indicates that FTser5ala has not been
retained in the appropriate site within the Golgi but moved to a more
distal compartment. By contrast, the ftGT is retained in the original FT subcompartment and adds Gal
(1,3)Gal to NAcLac (high IB4 levels), and therefore there is less acceptor remaining for the FTser5ala that
has shifted to a later subcompartment (reduced UEA-1 levels). As
mutations of the other amino acids did not affect UEA-1 staining, it is
clear that Ser5 is the most crucial amino acid for
functional localization in COS-7 cells.
An Hydroxyl Group at Amino Acid Position 5 Is the Essential
Component of FT Localization--
The S5A mutation had a crucial
effect on FT function although Ser and Ala differ by an hydroxyl group.
To determine whether this hydroxyl group was the essential element in
FT localization, Thr and Tyr (which also contain an hydroxyl group)
were also substituted at position 5. FT and FTser5tyr and FTser5thr
were cotransfected with GT, and the surface expression of
Gal
(1,3)Gal and H substance on COS-7 cells was determined (Fig.
5). When FT was transfected, 60% of
cells were stained with UEA-1 (Fig. 5A), and when GT was transfected 60% of cells were stained with IB4 (Fig. 5B).
When FT and GT were coexpressed the level of UEA-1 staining remained at
60% (Fig. 5C), and the level of IB4 staining was reduced to 20% (Fig. 5D). As shown above, FTser5ala was cotransfected
with GT, and the level of staining for both UEA-1 (Fig. 5E)
and IB4 (Fig. 5F) was 60%, showing again that the mutant
lacking the hydroxyl group could no longer compete with GT. However,
coexpression of FTser5tyr or FTser5thr (both containing an hydroxyl
group) with GT gave high levels of staining (60% of cells) with UEA-1
(Fig. 5, G and I, respectively) but not IB4 (Fig.
5, H and J, respectively, 20% of cells)
showing that an hydroxyl group at position 5 is important for the
ability of FT to compete with GT. This was confirmed using FTser5phe
and GT in cotransfections that gave similar IB4 and UEA-1 staining
patterns to FTser5ala and GT (data not shown), demonstrating that the
hydroxyl group rather than the ring structure of Tyr is important for
FT localization.

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Fig. 5.
Importance of the hydroxyl group to the
ability of FT to compete with GT for acceptor in COS-7 cells.
COS-7 cells were transfected with GT and FT mutants and then
surface stained with IB4-FITC (panels B, D,
F, H, and J) or UEA-1 (A,
C, E, G, and I). COS-7
cells were transfected with the following : A, FT + vector;
B, GT + vector; C, FT + GT; D, FT + GT; E, FTser5ala + GT; F, FTser5ala + GT;
G, FTser5tyr + GT; H, FTser5tyr + GT;
I, FTser5thr + GT; and J, FTser5thr + GT.
WT, wild-type.
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DISCUSSION |
The addition of carbohydrate moieties to proteins
and lipids occurs in a specific order, however the current models of
resident Golgi protein localization do not adequately describe
the mechanism of retention and localization of the
glycosyltransferases that perform the glycosylation. Here we
analyzed the involvement of the cytoplasmic tail of FT in
localization in the Golgi. When FT and GT are coexpressed, FT
first contacts the NAcLac acceptor and utilizes it prior to
GT, leaving little acceptor for GT, which results in a
reduction in Gal
(1,3)Gal on the cell surface (16, 17).
Removal of the cytoplasmic tail of FT reversed the ability of
FT to compete with GT, and Gal
(1,3)Gal expression was restored (Fig.
2). In addition, in the absence of the tail, GT utilized NAcLac
preferentially, which reduced the expression of H substance on the cell
surface (Fig. 2). Thus the cytoplasmic tail of FT is necessary for the
localization of the enzyme in the Golgi so that it can perform its
function with respect to competition with GT (17). Having shown that
the FT tail was important for function, we directly demonstrated that
after removal of the cytoplasmic tail, the intracellular distribution
of FT was altered in COS-7 cells. When examined by confocal microscopy,
FT was localized to the Golgi (Fig. 3), as also shown by others (15).
In contrast, FT without a cytoplasmic tail (FT
cyt) had a different
distribution and had moved to the TGN. Removal of the cytoplasmic tail
relocated FT from the Golgi to the TGN, and therefore the tail must be
important in the subcellular localization of FT. Analysis of the
8-amino acid cytoplasmic tail of FT showed that amino acids 3-7 were
involved in the localization (Fig. 4), and the serine at position 5 appeared to be the most critical. Furthermore, molecular replacement of Ser5 with threonine or tyrosine showed that the hydroxyl
residue is important for the correct localization and function (Fig.
5), whereas amino acids having the same side chains but lacking
hydroxyl groups (Ala and Phe) did not correctly localize. Thus, the
functional localization of FT involves the sequence in the cytoplasmic
tail, especially the hydroxyl at position 5.
It has been suggested that the lack of consensus sequences in the
glycosyltransferases (except for a common peptide stretch that has not
been assigned a function (23)) precludes the possibility of a specific
retention signal in the polypeptide sequence of the enzymes. However,
as presented here, removing the cytoplasmic tail of FT was sufficient
to alter the position of FT (Fig. 3) presumably by affecting molecular
interactions in the cytoplasm that localize it to a subcompartment of
the Golgi. In these interactions, the hydroxyl group of
Ser5 is important presumably by either formation of a
hydrogen bond or phosphorylation.
These findings of the importance of the cytoplasmic tail appear to be
different from other theories in terms of the localizing domain of the
enzyme but are compatible with current models of Golgi localization.
The kin-recognition model is based on the aggregation of homo- and
heterodimers into large complexes because of the hydrophobic
transmembrane domain of trans Golgi enzymes (9) or to the luminal
domain of the medial Golgi enzymes mannosidase II and
N-acetylglucosaminyltransferase I (4, 24). However, although aggregation may contribute to retention, it is not clear how
this aggregation could contribute to Golgi localization to a specific
site. Our data are consistent with the kin-recognition model, as
aggregation may be a prerequisite for the interaction of the FT
cytoplasmic tail with some localizing agent on the cytoplasmic surface
of the Golgi such as glycosyltransferase receptors or with a Golgi
matrix (25). Nonreducing SDS polyacrylamide gel electrophoresis showed
that FT is present as dimeric and larger molecular weight forms,
possibly tetrameric (data not shown); others have found it as a dimer
(15). This raises the possibility that multiple cytoplasmic tails
brought close together by dimer formation may be important for localization.
The second, lipid bilayer model (6) is reliant upon the general
property of hydrophobicity and length of the transmembrane domains
rather than upon specific sequences in the transmembrane domain for
retention within the Golgi; e.g. the shorter
17-amino acid transmembrane of sialyltranferase would not be able to
span the membranes of post-Golgi and TGN compartments (6). By removing the cytoplasmic tail of FT and leaving the transmembrane intact, we
have shown that FT moves to the TGN, which is not the Golgi subcompartment that normally allows preferential utilization of NAcLac.
This is consistent with the lipid bilayer model, as the transmembrane
domain may retain the enzyme and play a role as an anchor, without
which the transferase would be secreted. As we have not observed the
"tailless" version of FT in any vesicular structures or on the cell
surface, we presume it is the length and hydrophobicity of the
transmembrane domain of FT that leads to its retention in the TGN. Thus
the characteristics of the transmembrane domain are such that FT cannot
be transported beyond the TGN, whereas the tail acts to keep it in a
specific site in the Golgi.
Thus, it is likely that a number of different factors dictate the
precise localization of glycosyltransferases. Aggregation of the
enzymes and their transmembrane domains may be involved in general
Golgi retention, whereas retention sequences in glycosyltransferases may allow localization in specific functional subcompartments of the
Golgi. The concept of a second glycosyltransferase signal for specific
functional localization has also recently been suggested by others (26)
who carefully analyzed the biosynthetic products of
glycosyltransferases in vivo so that changes in their
localization leading to changes in product could be monitored.
The cytoplasmic tail is part of the general flanking sequence of the
transmembrane domain along with the stem region in the luminal domain,
which has been considered important for localization of Type II
membrane proteins (9-14). This study shows that the role of the
cytoplasmic tail of FT in Golgi localization is far more substantial
than previously thought and that there are only a few amino acids
involved in this phenomenon. The nature of the molecular interaction of
the FT tail with the cytoplasmic surface of the Golgi remains to be
determined, especially in light of recent findings of the importance of
the cytoplasmic portions of giantin (27) in Golgi localization.