(Received for publication, March 27, 1995; and in revised form, May 31, 1995)
From the
Glycosylphosphatidylinositol (GPI) anchors of the yeast Saccharomyces cerevisiae have been reported to contain three
different types of side chains attached to the 1,2-linked mannose
of the conserved
protein-ethanolamine-PO
-Man
1,2Man
1,6Man
1,4GlcNH
-inositol
glycan core. The possible side chains are Man
1,2- or
Man
1,2Man
1,2- or Man
1,3Man
1,2- (Fankhauser, C.,
Homan, S. W., Thomas Oates, J. E., McConville, M. J., Desponds, C.,
Conzelmann, A., and Ferguson, M. A.(1993) J. Biol. Chem. 268,
26365-26374). To determine in what subcellular compartment these
side chains are made, we metabolically labeled GPI-anchored membrane
proteins with myo-[2-
H]inositol in
secretion mutants blocked at various stages of the secretory pathway
and analyzed the anchor structure of the labeled glycoproteins. When
the exit of vesicles from the endoplasmic reticulum or entry into the cis-Golgi were blocked in sec12 or sec18 cells, all anchors contained a side chain consisting of a single
1,2-linked mannose. GPI proteins trapped in the cis-Golgi
of sec7 contained Man
1,3Man
1,2- but no
Man
1,2Man
1,2- side chains. Mutants blocked at later stages of
the secretory pathway made increased amounts of side chains containing
two mannoses. Man
1,2Man
1,2- and Man
1,3Man
1,2- side
chains were preferentially associated with ceramide- and
diacylglycerol-containing GPI anchors, respectively. Mnn1, mnn2,
mnn3, mnn5, and mnt1(=kre2),i.e. mutants
which lack or down-regulate 1,2- and 1,3-mannosyltransferases used in
the elongation of N- and O-glycans in the Golgi, add
the fifth mannose to GPI anchors normally. The same conclusion was
reached through the analysis of deletion mutants in KTR1, KTR2,
KTR3, KTR4, and YUR1 which all are open reading frames
with high homology to MNT1. Mutants deficient in the Golgi
elongation of N-glycans such as anp1, van1, mnn9 are
deficient in the maturation of the N-glycans of GPI-anchored
glycoproteins, but process the GPI anchor side chain normally. Data are
consistent with the idea that the fourth mannose is added to proteins
as part of the anchor precursor glycolipid in the endoplasmic
reticulum, whereas the fifth mannose is added by not yet identified
1,3- and
1,2-mannosyltransferases located in the Golgi
apparatus.
The addition of glycosylphosphatidylinositol (GPI) ()anchors to the carboxyl terminus of newly synthesized
polypeptides occurs as an early post-translational modification of
proteins entering the secretory pathway(1, 2) . While
the core carbohydrate structure linking the protein to the lipid moiety (Fig. 1) is conserved throughout eukaryotic evolution, many
organisms attach additional sugars and/or other groups to this core.
All GPI anchors of Saccharomyces cerevisiae contain a fourth
mannose residue (M4, Fig. 1) attached to the
1,2-linked mannose of the glycan core and part of yeast GPI
anchors also contain a fifth mannose (M5) which is linked
either
1,2 or
1,3 to M4 (3) . It is unknown if a
given protein is made with several different kinds of side chains or if
each protein is made with only one kind. The presence of a fourth,
1,2-linked mannose has also been found as a species- and
tissue-specific modification in mammalian GPI
anchors(4, 5) . Recently, the same
1,2-linked
mannose has also been found in Dictyostelium discoideum(6) and on a GPI glycolipid made by merozoites of Plasmodium falciparum(7) . Here we undertook to
exploit the well defined secretion and glycosylation mutants of S. cerevisiae in order to investigate the subcellular
localization and identity of the mannosyltransferases involved in the
biosynthesis of the mannose side chains of yeast GPI anchors.
Figure 1:
Glycosylphosphatidylinositol anchor
structures of yeast proteins. The scheme outlines the structural
variants found in S. cerevisiae(3) . The mannoses are
annotated by M1-M5. The majority of GPI anchors of wild type
cells contain only M1 to M4. A fifth mannose (M5) is present
only on part of the anchors and is linked either 1,2 or
1,3
to M4 (3) . The sites of cleavage obtained by HF
dephosphorylation and phosphatidylinositol-specific phospholipase C (PI-PLC) are indicated. Arrows point toward the
linkages which can be hydrolyzed by the
1,2-linkage-specific
exomannosidase from ASAM. EtNH, ethanolamine; R,
alkyl chain.
Figure 2:
Sec18 cells were radiolabeled with myo-[2-H]inositol at 24 °C (panels A and B) or 37 °C (panels C and D). Glycopeptides were generated and either left untreated (panels A and C) or treated with jack bean
-mannosidase (JBAM) (panels B and D).
Subsequently all glycopeptides were dephosphorylated with hydrofluoric
acid (HF), resulting fragments were N-acetylated and sized by
paper chromatography and scintillation counting of 1-cm wide paper
strips. M3-M5 indicate the migrations of radiolabeled
Man
-GlcNAc-Ins to Man
-GlcNAc-Ins standard
oligosaccharides (18) run in parallel on the same paper.
Labeling temperatures and the sequential order of treatments are
summarized at the top of each panel.
Figure 3: Anchor glycopeptides were prepared from radiolabeled X2180 cells, were divided into 4 equal aliquots, and were subjected to sequential treatments in the order indicated on top of each panel. N-Ac, N-acetylation. Resulting fragments were sized by paper chromatography. Recoveries of counts/min were close to 100% in all treatments.
The slower migrating peak was assumed to represent a
fragment with a fifth mannose (M5, Fig. 1) which is
predicted by the previous analysis of yeast anchors(3) . Its
identity was confirmed by the following observations: (i) when isolated
from a preparative paper chromatogram and treated with jack bean
-mannosidase (JBAM), the slower migrating peak yielded a fragment
comigrating in thin layer chromatography with GlcNAc-Ins, not Ins (not
shown). (ii) When JBAM treatment of total anchor peptides was done before HF treatment, counts were quantitatively recovered in a
peak comigrating with the Man
-GlcNAc-Ins standard
indicating that three mannoses were protected by an HF-sensitive group
also in this slower migrating peak (Fig. 3, panel C).
(iii) The HF fragment migrated much slower if N-acetylation
was omitted, thus indicating the presence of an amino group which is
typically found on the glucosamine of GPIs (Fig. 3, panel
A). (iv) Treatment of the total of anchor peptides with
-mannosidase from A. saitoi (ASAM) produced a
fragment comigrating with Man
-GlcNAc-Ins whereby part of
the material proved to be resistant (Fig. 3, panel D).
These findings strongly argue that this additional HF fragment
represents a GPI structure. On the basis of the previous analysis of
the GPI anchor of mature GPI proteins from the same strain
(X2180)(3) , it seems safe to assume that the slower migrating
peak represents a mixture of
Man
1,2Man
1,2Man
1,2Man
1,6Man
1,4GlcNAc
1,6Ins
and
Man
1,3Man
1,2Man
1,2Man
1,6Man
1,4GlcNAc
1,6Ins,
the latter being resistant to ASAM. There is some variability among
different wild type strains with regard to the fraction of GPI anchors
containing a fifth mannose (M5, Fig. 1, Tables II and
IV). It should be noted that the percentages obtained for wild type
cells represent the status of GPI anchors in the Golgi and/or in
post-Golgi compartments. This can be stated because, when analyzed by
SDS-polyacrylamide gel electrophoresis, ER forms of GPI proteins are no
more detectable after a 2-h pulse labeling with myo-[2-
H]inositol as was done in the
experiments described in here(23) . (ER forms of GPI proteins
have much lower molecular masses than more mature GPI proteins.)
In summary, ER forms of GPI-anchored proteins contain the same four mannoses as the candidate precursor lipids CP1 and CP2. Moreover, the absence of M5 in secretion mutants which block vesicular traffic between the ER and Golgi strongly suggests that the addition of M5 occurs in the Golgi.
To probe the
distribution of transferases involved in the addition of M5, we used sec7, a secretion mutant which blocks between early and mid
Golgi compartments (24) (Table 1). The disappearance of
1,2-linked but not
1,3-linked M5 in sec7 upon shift
to 37 °C strongly suggests that an
1,3-mannosyltransferase is
encountered by newly made GPI proteins already in the earliest Golgi
compartment lying proximal to the sec7 block whereas the
1,2-mannosyltransferase is localized in later Golgi compartments
lying beyond the sec7 block. While interpreting this result
one should keep in mind that significant amounts of enzymes destined
for a distal compartment will accumulate proximally to a secretory
block that is maintained for some time. This implies that during a
prolonged secretory arrest in sec7, the cis-Golgi
compartment might actually take on characteristics of a mid or trans-Golgi compartment and carry out distal modifications.
Thus, the absence of
1,2-linked M5 in sec7 allows to
formally conclude that in wild type cells,
1,2-linked M5 is added
in the mid or trans-Golgi. On the other hand, in spite of the
persistence of
1,3-linked M5 in sec7, the corresponding
1,3-mannosyltransferase cannot be assigned unequivocally to the cis-Golgi since
1,3-mannosyltransferase might be a trans-Golgi enzyme which, if artificially retained proximal to
the sec7 block, assumes an active conformation. The trans-Golgi localization of the
1,3-mannosyltransferase
may appear less likely since the enzyme is not active if retained in
the ER proximal to a sec18 block and because the related
1,2-mannosyltransferase is not active if retained in the cis-Golgi.
In this context it should be noted that there is no
increase in 1,3-linked M5 in sec7 as compared to wild
type. Assuming that the corresponding
1,3-transferase is located
in the cis-Golgi also in wild type cells, this result suggests
that in wild type the exposure time of GPI proteins to
1,3-mannosyltransferase in the cis-Golgi is not limiting
or else that Sec7p is required to maintain the
1,3-mannosyltransferase of the cis-Golgi in a functional
state. Later secretion mutants (sec14 and sec1) seem
to enhance addition of M5 (Table 1), possibly because GPI
proteins remain for prolonged periods in contact with
1,3- and the
1,2-mannosyltransferases present in the later Golgi. It is
noteworthy that a block beyond the Golgi (sec1) induces an
increase in both
1,2- and
1,3-linked M5, whereas the
intra-Golgi block of sec14 results in a decrease of
1,2-
but a compensatory increase in
1,3-linked M5 (Table 1).
Since the sec14 block is supposed to be distal to the sec7 block, this might indicate that the sec14 block renders
the access of GPI proteins to the
1,2-mannosyltransferase
difficult and that part of the
1,2-transferase is located in a
very late Golgi compartment. Alternatively, the sec14 block
might delay transition of GPI proteins through the early and middle
Golgi, thus increasing the time of exposure of the anchors to
1,3-mannosyltransferase(s) so that less substrate would be left
for the
1,2-mannosyltransferase.
Figure 4:
1.5 10
cells from
different insertional mutants in N-glycan elongation and
corresponding parental cells were labeled for 2 h with 20 µCi of myo-[2-
H]inositol at 24 °C. Proteins
were extracted and analyzed by SDS-polyacrylamide gel
electrophoresis/fluorography. Exposure was for 1 week. The GPI anchor
maturation of these mutants is summarized in Table 4.
As for many other glycan structures, the exact role of the
mannose side chain on GPI anchors is presently not well understood.
Nevertheless, single mannose side chains (M4) have been found in
mammals, D. discoideum, P. falciparum, and S. cerevisiae and therefore, the possibility to add
M4 seems to have been maintained during evolution in several phyla. On
the other hand, the GPI biosynthesis machinery, at least of mammals and
trypanosomes, does not require the presence of M4, since most mammalian
and trypanosomal cells do not contain this residue either on the
complete precursor lipids or on the GPI proteins. In contrast to M4, M5
residues have only been described in S. cerevisiae. In various
wild type strains, M5 residues are present in 18-32% of anchors,
and the ratio of 1,2- versus
1,3-linked mannose
varies from 1:4 to 2.5:1, depending on the strain (Table 2).
The identity of the mannosyltransferases involved in the addition of
M4 and M5 are unclear at the moment. The general experience in
glycobiosynthesis is that each kind of linkage is achieved by a
different glycosyltransferase. Since M4, as M3, is 1,2-linked, it
is conceivable that the same mannosyltransferase is responsible for the
addition of both M3 and M4. On the other hand, the M5 adding
mannosyltransferases are definitely different from the ones that add M4
since, according to our data with secretion mutants, they reside in the
Golgi whereas the M4 addition must occur in the ER. Our data show that
none of a panel of cloned Golgi mannosyltransferases or genes
regulating such transferases is essential for the addition of
1,2-linked or
1,3-linked M5. The slight reduction in
1,3-linked M5 observed in mnn3, ktr3, and ktr4 mutants might be taken as an indication that all of these enzymes
are involved in the addition of
1,3-linked M5, but this would be
against the general ``one linkage-one enzyme'' rule mentioned
before. Also, this seems unlikely in view of the fact that KTR3 shows much less homology with KTR4 than with KTR1 which latter is without influence on the addition of M5 (Table 2)(12) . Thus, although we cannot exclude that
redundant enzymes are responsible for the addition of M5, it seems
more likely that M5 addition is due to the presence of some other, yet
unknown Golgi mannosyltransferases.
The GPI anchor peptides analyzed here were purified over octyl-Sepharose and hence contain a lipid moiety. We conclude that the M5-transferases get access to M4 without any need for previous removal of the lipid moiety. This is in agreement with the idea that the glycan part of GPIs can assume a relatively extended configuration and form a broad platform between protein and lipid(40) . Yet, we routinely find that 10-20% of the anchor peptides eluted from ConA-Sepharose do not bind to octyl-Sepharose. Indeed it has recently been reported that for some proteins the GPI anchor represents a necessary and sufficient signal for their incorporation into the cell wall and that upon arrival at the plasma membrane part of the GPI anchor including the lipid moiety is removed(39, 41, 42) . Having restricted our analysis to lipid-containing anchors, it is obvious that the possible further additions of glycans onto the GPI core structure or onto the GPI side chain during this incorporation process would have escaped detection.
Through analysis of N-glycan structures
of glycoproteins accumulating in sec7, sec14, sec18, and sec23, of -factor maturation events in these secretion
mutants and through subcellular fractionation studies, the yeast Golgi
could be divided into two distinct early (cis) compartments containing
1,6-mannosyltransferases for the elongation of N-glycans,
a later (mid) compartment containing the N- and O-glycan elongating
1,3-mannosyltransferase coded for by MNN1, and an even later (trans) compartment containing the
processing protease coded for by KEX2(24, 25, 26, 27) . The
presence of
1,3-linked or
1,2-linked M5 residues on GPI
anchors can now serve as an alternative means for tracking GPI proteins
and their vesicular flow through the Golgi. The presence of
1,2-linked M5 indicates that a GPI protein has reached or passed
the later Golgi compartments whereas the addition of
1,3-linked M5
in sec7 cells indicates that a GPI protein has reached the cis-Golgi. Subcellular fractionation studies will be required
to decide whether the presence of an
1,3-mannosyltransferase is a
distinguishing feature of the cis-Golgi also in wild type
cells. It should be noted that early and late Golgi modifications of
GPI anchors are independent or even mutually exclusive events, whereas
in the N-glycan elongation, addition of
1,6-mannose in
the early Golgi is a prerequisite for the addition of
1,3-linked
mannose in the later Golgi.
Base-sensitive anchors have a tendency
to receive 1,3-linked M5 whereas base-resistant anchors
preferentially are provided with an
1,2-linked M5. The bias of
1,3-linked M5 for base-sensitive anchors can be interpreted in
several alternative ways which at present are not easy to distinguish
experimentally. (i)
1,3-Mannosyltransferases might prefer
diacylglycerol-based anchors over ceramide-based ones, and the inverse
might be true for the
1,2-mannosyltransferase. (ii) Since lipid
moieties of GPI anchors might still get exchanged in the
Golgi(17) , the Golgi lipid exchange enzyme might introduce
ceramides preferentially on anchors with
1,2-linked M5. (iii) GPI
proteins might get sorted according to their lipid domain, and
diacylglycerol-based GPI proteins might have more access to the
1,3-mannosyltransferase of the early Golgi whereas ceramide-based
ones have better access to the
1,2-mannosyltransferase of the late
Golgi. (iv) Diacylglycerol-based GPI proteins might transit more slowly
through the Golgi than ceramide-based ones so that the cis- or
mid Golgi
1,3-mannosyltransferases have more time to attach a M5
and that the
1,2-mannosyltransferase of the late Golgi cannot find
any suitable substrate any more. Enzyme specificities invoked in i and
ii are rendered less likely by the high amounts of
1,3-linked M5
found on ceramide-based anchors of mnn9. This latter finding
clearly shows that either
1,3-mannosyltransferase can act on
ceramide-based anchors or that the lipid exchange enzyme can act on
anchors with
1,3-linked M5. Sorting of GPI proteins according to
their lipid moiety (possibilities iii and iv) would represent a novel
mechanism. However, it has previously been demonstrated that the
vesicular transport of yeast GPI proteins from ER to Golgi is dependent
on ceramide biosynthesis whereas the vesicular flow of membrane
proteins containing a hydrophobic, membrane spanning sequence is not
dependent on ceramide biosynthesis(43) .
The studies with mutants such as mnn9, van1, anp1, and erd1 demonstrate that their deficiency only abolishes the capacity of N-glycan elongation but does not eliminate other glycosylation events of the Golgi such as addition of M5 to GPI proteins. Mannosylation of inositolphosphoceramides seems to be intact, since the pattern of inositolphosphoceramides and the mannosylated forms thereof are normal in all of these mutants (not shown). Further studies will be required to delineate the primary event leading to this specific deficiency in N-elongation.