(Received for publication, May 27, 1994; and in revised form, October 20, 1994)
From the
FP21 is a glycoprotein which, when tracked by radioactivity in
its fucosyl moiety, was previously detected in the cytosol of Dictyostelium cells after cell fractionation. This
compartmentalization is confirmed by SDS-polyacrylamide gel
electrophoresis/Western blotting of cell fractions using three
different antibodies. Although a substantial fraction of FP21 is also
detected in the particulate fraction using these new antibodies,
particulate FP21 is released by disrupting protein-protein
interactions, but not membrane disruption. Since purified FP21 is
susceptible to aggregation, and purified nuclei do not contain FP21,
particulate FP21 is also part of the cytosol. Additional compositional
and structural information provides strong evidence that FP21 does not
at any time traverse the rough endoplasmic reticulum. First, cDNAs
spanning the entire coding region of the FP21 gene predict no
hydrophobic motifs expected to promote membrane insertion, but do
predict an NH-terminal coiled coil domain which could
explain aggregation. Second, monosaccharide composition analysis of the
predominant glycoform of FP21 yields 2 mol of galactose, 1 mol of
xylose, and 1 mol of fucose/mol of polypeptide; FP21 from a
fucosylation-defective mutant contains 1 additional mol of xylose in
place of fucose. Thus the N-glycosylation sequon present in
FP21 is not utilized by oligosaccharyl transferase, which resides in
the rough endoplasmic reticulum. These findings indicate that nascent
FP21 remains in the cytosol after synthesis and is therefore
glycosylated by unusual cytosolic xylosyl-, galactosyl-, and
fucosyltransferases.
An increasing number of proteins which reside in cytosolic and
nucleoplasmic compartments of the cell are becoming recognized as
glycosylated with a single GlcNAc residue(1, 2) ,
lending credence to a sizable body of earlier data indirectly
suggesting glycosylation of multiple cytosolic and nucleoplasmic
proteins(3) . Recently, direct chemical evidence has also been
obtained for the existence of oligosaccharides on certain proteins of
the cytosol(4, 5) . Since the cytosol is the cellular
compartment of protein translation, the simplest mechanism for
glycosylating these proteins would involve cytosolic
glycosyltransferases, to join several cytosolic glycosyltransferases
involved in synthesis of, e.g. dolichol sugars (6) and
glycogen(7) . However, since at present all known protein
glycosyltransferases which have been cloned are type two membrane
proteins with their active sites oriented toward the lumen of either
the rER, ()the Golgi complex, or a topologically continuous
space(8, 9) , an alternative model is that the
cytosolic protein translocates into and then back out of a conventional
glycosylation compartment.
Consistent with the model that cytosolic
glycoproteins are glycosylated in the cytosol, a uridine
diphospho-N-acetylglucosamine:polypeptide
-N-acetylglucosaminyltransferase activity has been
purified to apparent homogeneity from a cytosolic preparation (10) of the liver. This enzyme catalyzes the glycosidic linkage
of GlcNAc to the hydroxyl of serine or threonine of cytosolic and
nuclear proteins. The existence of larger tri- and tetrasaccharide
modifications suggests that there are additional cytosolic
glycosyltransferases capable of synthesizing a variety of interesting
oligosaccharide structures, if the compartmentalization of the O-GlcNAc transferase is conserved.
One such cytosolic glycoprotein bearing an oligosaccharide is FP21 from the simple eukaryotic cellular slime mold Dictyostelium discoideum. FP21 possesses a tetra- or pentasaccharide whose polypeptide linkage is susceptible to mild alkaline degradation(5) . Characteristics of the FP21 polypeptide reported here strongly indicate that it is glycosylated in the cytosolic compartment and, using a sensitive micromethod, we find that it contains Gal, Xyl, and Fuc and lacks GlcNAc and GalNAc. This suggests that the Dictyostelium cytosol contains novel galactosyl and xylosyltransferases in addition to the FP21-fucosyltransferase already detected there (5) and that these transferases comprise a glycosylation pathway independent of the GlcNAc modification pathway cited above.
mAb 3F9 IgG was purified from
ascites by a caprylic acid/(NH)
S0
differential precipitation method(17) , and a 15 mg/ml
solution was coupled to CNBr-activated Sepharose (Pharmacia Biotech
Inc.) according to the manufacturer's instructions. Purified
rabbit IgG (Sigma) was coupled in a similar manner.
Additional mAbs were developed after immunization of mice with FP21 purified from strain Ax3 according to the protocol described below. Two of these antibodies, 3F5 and 3D11, show similar binding to wild-type and mutant FP21 in Western blots of cell extracts, compared with mAb 3F9.
An
antiserum was prepared against a synthetic peptide consisting of
CQGDDKKDEKRLDDIP, which corresponds to residues 68-82 of FP21
(see Fig. 5) plus an NH-terminal Cys for coupling.
The peptide was coupled to maleimide-activated keyhole limpet
hemocyanin and ovalbumin using the Imject(TM) Activated Immunogen
Conjugation Kit (Pierce). Rabbits were immunized with the keyhole
limpet hemocyanin conjugate, and sera were titered using an ELISA assay
based on the ovalbumin conjugate. Anti-FP21-(68-82) from rabbit
L97 gave a 50% reaction in the ELISA assay at a dilution of 1:7500. The
antiserum was affinity-purified on a column prepared by conjugating
approximately 5 mg of the original peptide to 2 ml of
2-nitro-5-thiobenzoate-thiol agarose (Pierce), according to the
manufacturer's protocol. After acid elution and neutralization,
anti-FP21-(68-82) showed a specificity for FP21, based on Western
blots blocked in milk, which was similar to the monoclonal antibodies.
Figure 5:
FP21 cDNA sequence and translation of the
single, long open reading frame. The sequence of the overall cDNA group
of overlapping clones assembled from primer-dependent amplification
products from the ZAP cDNA library, and partially confirmed by
corresponding amplification products from genomic DNA, is shown,
together with translation of the single long open reading frame. The underlined amino acid sequence from residues 4-38 was
previously determined by Edman degradation of the intact purified
protein(5) . The underlined amino acid sequence from
residues 31-43 was obtained from the M
11,000 CNBr fragment described in Table 1. A synthetic
peptide encompassing the underlined sequence, including
residues 68-82, was found to induce an antiserum which, after
affinity purification against the peptide, was monospecific for FP21
(data not shown). The underlined nucleotide sequences
represent two consensus polyadenylation
signals.
A Triton X-100-resistant cytoskeletal fraction, and associated soluble fraction, were prepared as described previously(13) . An actomyosin fraction was prepared(14) , as modified according to (15) , except that cells were lysed by forced passage through a 5-µm pore diameter nuclepore filter.
Nuclear fractions
were prepared as described(16) , using both the digitonin and
Nonidet P-40 methods. Nuclei were detected by staining with 30
µg/ml propidium iodide, diluted from a 100 stock solution
in water, and visualized epifluorescently through a rhodamine filter
channel. To improve the purity of nuclei, nuclear preparations were
layered on a 1.0-2.0 M linear gradient of sucrose in
nuclear lysis buffer and centrifuged in a swinging bucket rotor at
70,000
g for 20 min. Nuclei were most enriched in
turbid bands near the bottom of the gradient, well separated from other
organelles, but still partially contaminated by intact cells.
cDNA was
amplified by a PCR protocol (cycle profile: 1 min, 94°; 1 min,
49°; 5 min, 72°) using Taq polymerase (Promega) in 3
mM MgCl. PCR-derived DNA fragments were gel
purified and subcloned into either pBluescript (Stratagene, La Jolla,
CA) or pGEM-T (Promega). Dideoxy sequencing was performed at an
Interdisciplinary Center for Biotechnology Research core laboratory at
the University of Florida on an ABI 373a DNA sequencer system. All
regions reported were sequenced two to six times in each direction from
two or more different PCR amplifications.
Figure 1:
FP21 is found
in S100 and P100 fractions at different stages of the life cycle. Lanes a-f, growth phase cells grown either on K.
aerogenes (lanes a and b) or axenically (lanes c and d), or developed to the preculmination
(18 h) stage (lanes e and f), were resolved by
SDS-PAGE and analyzed by Western blotting using mAb 3F9. The use of
hemoglobin as the blocking agent led to a higher level of nonspecific
binding than that observed using 5% non-fat dry milk (see Fig. 2and Fig. 3). Lanes g-j, S100 (lanes g and h) and P100 (lanes i and j) fractions were prepared from axenically grown vegetative
cells and analyzed in like fashion. The normal strain Ax3 (lanes a,
c, e, g, and i) and fucosylation-defective strain HL250 (lanes b, d, f, h, and j) samples are arranged in
pairwise fashion in alternate lanes. Each lane contains 100 µg
protein. The region of the gel from approximately M 10,000-35,000 is shown. The position of FP21 is indicated
by a double bar, since its apparent M
differs slightly between the two
strains.
Figure 2:
Changes in ionic strength or pH release
FP21 from the P100 (particulate) fraction. The P100 fraction of
axenically grown vegetative Ax3 cells was resuspended either in
original lysis buffer, 0.25 M sucrose, 50 mM Tris-HCl, pH 7.4 (lanes a and b), or in 0.1 M NaC0
, pH 11.5 (lane c), 1 M NaCl, 10 mM Na
EDTA, 50 mM Tris-HCl, pH 7.4 (lane d), 10 mM Na
EDTA-Na0H, pH 7.4 (lane e), or 0.5% Triton
X-100, 50 mM Tris-HCl, pH 7.4 (lane f). Samples in lanes b-f were probe-sonicated to facilitate
resuspension. To determine whether FP21 was released from the
particulate fraction by these treatments, the samples were
recentrifuged. The resulting P100 (upper panel: new pellet)
and S100 (lower panel: new supt) fractions were examined by
SDS-PAGE and Western blotting with mAb 3F9.
Figure 3:
Bulk soluble FP21 is not accessible to in vitro fucosylation. An S100 fraction from axenically grown
HL250 vegetative cells was labeled with
GDP-[H]Fuc and precipitated with progressively
higher concentrations of (NH
)
S0
.
Successive cuts, listed as percent saturation, were collected by
centrifugation, dissolved, and dialyzed. The distribution of total S100
protein among cuts is given in the bar graph (light
shading). Equal amounts of total protein from each cut were
resolved by SDS-PAGE and analyzed either by Western blotting with mAb
3F9 for FP21 (shown in lower panel) or by counting gel slices
containing FP21 (bar graph, dark shading)(5) . Whereas
FP21 is fairly equally distributed over all cuts on a per protein
basis, only a small fraction of total FP21 (<5%) resides in the
70-80% (NH
)
S0
fraction,
because <5% of total protein precipitates in this fraction. In
contrast, >80% of FP21 which can be fucosylated in vitro precipitates in this fraction, suggesting the bulk of FP21 is
protected from in vitro fucosylation.
In
order to demonstrate that mAb 3F9 reacts with FP21, a mAb 3F9 affinity
column was prepared and used to purify
[H]fucosyl-FP21, prepared by metabolic labeling
of strain Ax3 vegetative cells with [
H]fucose, or
by labeling FP21 in a strain HL250 S100 preparation with
GDP-[
H]fucose(5) . In each case,
radioactive FP21 was recovered from the S100 fraction at >70% yield,
whereas <5% of total protein was bound to the column. Thus mAb 3F9
binds to FP21 and is highly specific for FP21 with respect to total
cell protein.
mAb 3F9 was used to assay FP21 at different stages of development. FP21 was present after 18 h of development (the preculmination stage), and axenically grown vegetative cells at a similar level, and was also present in bacterially grown vegetative cells (Fig. 1, lanes a, c, and e). A similar result was found in mutant strain HL250 (Fig. 1, lanes b, d, and f), except that a strongly reactive protein of a smaller size was also detected reproducibly with mAb 3F9 only in bacterially grown vegetative HL250 cells (Fig. 1, lane b). The identity of this band is unknown.
To determine the
compartmentalization of FP21, cells were filter-lysed in sucrose/Tris
buffer and separated by centrifugation into S100 (cytosolic) and P100
(total particulate) fractions. mAb 3F9 detected FP21 in both the S100 (Fig. 1, lanes g and h), where it was
originally found(5) , and at a similar but slightly lower
level, on a per total protein basis, in the P100 (Fig. 1, lanes i and j). Although previously
[H]fucosyl-FP21 was not detected in the P100
fraction (5) , as if P100-associated FP21 is not fucosylated,
the M
difference between wild-type and mutant
isoforms of FP21 was conserved in the P100 fraction. This discrepancy
could be explained if the fucose turned over more rapidly, in the cell,
on FP21 in the cytosol relative to the insoluble form. To evaluate the
nature of the association of FP21 with the particulate fraction,
aliquots of the P100 were resuspended in various solubilizing agents
and recentrifuged, and the new pellets and supernatants were compared
by SDS-PAGE/Western blotting. Lane a (Fig. 2) shows
that almost half of the P100-bound FP21 was eluted by simply washing in
the same sucrose/Tris buffer. The remaining P100-bound FP21 was not
eluted by sonication (lane b), indicating that particulate
FP21 is not a soluble protein trapped within a vesicle. In contrast,
high pH (lane c), high salt/EDTA (lane d), and low
salt/EDTA (lane e) each nearly completely released FP21 into
the supernatant. Finally, non-ionic detergent stabilized FP21's
association with the pellet (lane f). Similar results were
observed for HL250-derived FP21 (not shown). If FP21 is associated with
membranes, it must be external and peripheral.
To address the possibility that soluble FP21 might be particulate-associated in the intact cell in a manner which is sensitive to the conditions of cell lysis, cells were lysed in buffers which have been optimized for the preservation of cytoskeletal components. FP21 was not detected in a Triton X-100 cytoskeletal fraction which enriches for actomyosin or in a purified actomyosin fraction (data not shown), prepared as described under ``Experimental Procedures.'' Finally, FP21 was not present in either of two nuclear fractions prepared as described under ``Experimental Procedures'' (data not shown). If FP21 is concentrated in the nucleus of intact cells, it must be weakly bound, allowing it to diffuse out during the isolation procedure. Because purified FP21 tends to self-aggregate (see below), it is likely that particulate FP21 consists simply of aggregates of FP21 which are sensitive to high pH, high salt/EDTA, or low salt/EDTA.
Since (NH)
SO
precipitation did
not enrich for bulk FP21, a new purification protocol was developed
which bypassed precipitation, retained DEAE ion exchange
chromatography, and employed a mAb 3F9 affinity column (see
``Experimental Procedures''). Fig. 4compares the
Coomassie Blue-stained SDS-PAGE profiles of the wild-type prerun (pr),
flow-through (ft), and acid-eluted fractions (1, 2, 3, 4, 5) from the
affinity column (after warming and centrifugation, see next paragraph),
in addition to acid eluted fractions (3, 4) from a
column containing mutant FP21. FP21 is the predominant protein in the
eluted fractions for both wild-type and mutant FP21.
Figure 4:
Purification and aggregation of FP21. FP21
was purified separately from the S100s of axenically grown Ax3 and
HL250 vegetative cells by DEAE-Sephadex chromatography and adsorbed
onto mAb 3F9 affinity columns. Each affinity column was eluted with 0.1 M glycine HCl, pH 2.5, and fractions were neutralized and
precentrifuged at 4 °C. After warming the supernatants to 22
°C, aggregated protein was collected by centrifugation at 13,000
g for 10 min and compared with remaining soluble
material by SDS-PAGE and staining with Coomassie Blue. Shown in the
first two lanes are the prerun (pr), flow-through (ft), followed by successive acid-eluted fractions (1-5) from the strain Ax3 sample, resolved into pelleted (left member of each pair) and soluble (right member
of each pair) subfractions. An M
ladder is shown
in the middle, consisting of, from top to bottom, M
values 66,200, 45,000, 36,000, 29,000, 24,000,
20,100, and 14,300, as marked in the right-hand margin. Shown
to the right of the M
ladder is a mixture
of fraction 4 from Ax3 and fraction 3 from HL250 (M for mix),
followed by the acid-eluted fractions (3 and 4) from
a parallel affinity column containing a sample from strain HL250.
Greatest sedimentability of FP21 is observed for the fractions with the
highest protein concentration, and differences between the two strains
in the contaminating minor proteins which cosediment with FP21, suggest
that their association is nonspecific.
Fig. 4also illustrates the tendency of FP21 to aggregate.
Protein recovered in the neutralized, acid-eluted fractions from the
mAb 3F9 column was observed to undergo rapid clouding upon warming to
23 °C, which was reversible upon returning to 0 °C. This
behavior was visible through at least five cycles of warming/cooling.
To determine which proteins were aggregating, after two cycles,
rewarmed fractions were centrifuged, and the pellet and the clear
supernatant for each fraction were electrophoresed in pairs (Fig. 4). FP21 was partially sedimentable, and when its
concentration exceeded approximately 50 µg/ml, the majority of FP21
was pelleted. A similar concentration dependence was observed for FP21
from both strains Ax3 and HL250. Although other proteins were also
sedimented, their involvement appears to be nonspecific, because each
is present in a lower quantity, and proteins of different apparent M were sedimented in the Ax3 and HL250
preparations. This behavior suggests that FP21 self-aggregates,
although it cannot be ruled out that other minor proteins contribute to
this process by a substoichiometric mechanism.
The M of gel-purified FP21 was determined independently by
MALDI-TOF-MS. As summarized in Table 1, the M
of normal FP21 averages 20,500 and mutant FP21 averages 19,800,
in close agreement with the M
differences
estimated by SDS-PAGE. The M
values of both
proteins appear heterodisperse, possibly due to the presence of SDS.
Alternatively, heterodispersity may reflect variability in the extent
of multiple posttranslational modifications.
To estimate the isoelectric point, a whole cell soluble (S100) fraction, and a mAb 3F9 column-purified FP21 fraction, were subjected to two-dimensional O'Farrell gel electrophoresis and either stained with Coomassie Blue, or silver, or Western-blotted with mAb 3F9 as described under ``Experimental Procedures.'' In both fractions, FP21 electrophoresed as a monodisperse spot with no evidence for multiple isoelectric isoforms, with an apparent pI of 5.5.
To confirm and
extend earlier data on the glycosylation of FP21, identical aliquots of
the above gel-purified preparation were spotted onto PVDF membranes in
parallel, hydrolyzed, and subjected to monosaccharide and amino acid
composition analysis. Separate elution programs were required to
optimize the separation of amino and neutral sugars, Xyl and Man, and
uronic acids. As summarized in Table 2, each mole of normal FP21
contains approximately 2 mol of Gal, 1 mol of Xyl and 1 mol of Fuc;
substoichiometric amounts of GlcNH and GalNH
were detectable at <0.2 mol; Glc could not be determined owing
to high background in the standard protein controls and the PVDF
blanks. No other peaks greater than 0.1 mol/mol polypeptide were
present in the chromatogram from 4-24-min retention time. Since
FP21 resolves into two peaks after DEAE-Sepharose chromatography, the
sugar composition of these two peaks was compared. The majority of FP21
(75-80%) is present in peak II, and material from this peak had
approximately the same molar ratios of Gal, Xyl, and Fuc, 2:1:1, as the
pooled material. A lower level of Glc (1 mol/mol Fuc) is detected,
presumably because these samples were acetone-precipitated rather than
blotted onto PVDF paper, which contains variable levels of Glc after
acid hydrolysis. In contrast, peak I contained Gal, Xyl, and Fuc in a
1:2:1 ratio, and Glc:Fuc was approximately 3:1. It is not known whether
Glc is covalently attached to FP21.
The amino acid composition (Table 3) of FP21 was consistent with the prediction from the FP21 cDNA sequence (see below), given that Met, Ser, and Thr are typically underestimated by the technique employed, and that Ala and Gly are typically overestimated, with high levels of Gly likely deriving from the SDS-PAGE purification step, which included glycine in the buffers. After the predicted composition was used to estimate the levels of amino acids not quantified chemically, FP21 was found to consist of about 3% carbohydrate, if glucose is absent.
The molar Fuc content of pooled peaks I and II of mutant FP21 was, as expected(5) , less than 0.05 (Table 2). The relative molar content of Xyl was increased to approximately 2. The amino acid composition of mutant FP21 was indiscernible from that of the normal form (Table 3).
No phosphate or sulfate could be detected under conditions of metabolic labeling that efficiently labeled other proteins (see ``Experimental Procedures''), including minor contaminating proteins which coeluted with FP21 from the mAb 3F9 column (data not shown).
The start of the long open reading frame is preceded by a 93-bp A/T-rich sequence and possesses a typical translation start context (31) . The long open reading frame contains an A/T content and codon usage typical of Dictyostelium coding regions(32) . The open reading frame ends with an ochre (TAA) codon, also typical for Dictyostelium, and is followed by about 15 bp of nucleotides having a coding region base composition, followed by 90 A/T-rich bp. This is followed by a 60-bp open reading frame with an A/T content typical of coding regions. This short, isolated open reading frame, flanked on both sides by A/T-rich sequences, is followed shortly by two AATAAA polyadenylation consensus signals within a 55 bp A/T rich 3` segment. The overall sequence contains 801 bp, in agreement with the length of the single hybridizing band observed by Northern blotting of total RNA (800 bp; see Fig. 6) taken from cells under two different nutritive conditions and two stages of development.
Figure 6: Northern blot of FP21 expression. Northern blot analysis of Ax3 RNA. Lanes marked 0 and 13 h contain total RNA from axenically grown vegetative cells (26 µg) and 13-h filter-developed cells (15 µg), respectively; the size of Dictyostelium rRNAs in kilobases are indicated on the left. The lane marked Lad (ladder) contains 5 µg of RNA standards with sizes in kilobases labeled as indicated. Left-hand panel, low-stringency wash; right-hand panel, subsequent high-stringency wash of the same blot. The probe was a random prime-labeled 0.5-kilobase fragment amplified with primers 12 and 16 from cDNA.
The
long open reading frame is concluded to encode FP21 for the following
reasons. 1) The message length predicted by the cDNA group of
overlapping clones is similar to the length of the mRNA determined by
Northern blotting (800 bp; see Fig. 6). The FP21 message is
present during development and during growth on either bacteria or
axenic medium (Fig. 6), consistent with the protein accumulation
data (Fig. 1). 2) The cDNA sequence correctly predicts the
40-amino acid sequence determined by Edman degradation of the isolated
protein. 3) The chemically determined amino acid composition of
purified FP21 is accurately predicted by the long open reading frame (Table 2). 4) The long open reading frame predicts a polypeptide
with a length of 162 amino acids and an M of
18,689, which accounts for about 91% of the M
estimated by MALDI-TOF mass spectrometry (Table 1). 5) The
nucleotide sequence predicts the NH
-terminal amino acid
sequence and the size of FP21's largest CNBr fragment (Table 1). 6) As a further test of the validity of the predicted
FP21 amino acid sequence, a rabbit antiserum was generated against a
synthetic peptide, spanning predicted residues 68-82 (see Fig. 5), which was covalently coupled to keyhole limpet
hemocyanin. Anti-FP21-(68-82) recognized both wild-type and
mutant glycoforms of purified FP21 after SDS-PAGE and Western blotting
at dilutions of greater than 1:5000. After affinity purification of the
antiserum against the peptide coupled to
2-nitro-5-thiobenzoate-thiol-agarose beads, the antibody was specific
for FP21 by Western blotting (data not shown).
The short, isolated
open reading frame, 3` to the long open reading frame and 5` to the
polyadenylation signal, is an unusual feature of 3`-untranslated RNA.
Several observations indicate that this feature was not an
amplification or recombination artifact. First, oligonucleotides which
hybridize with the short open reading frame (fp9, fp14, and fp16; Table 4) each, in pairwise combination with oligonucleotides
hybridizing with the long open reading frame (fp7, fp10, fp12, fp13,
and fp15), primed unique PCR products of identical and expected lengths
whether the template was 1) DNA from the ZAP cDNA library, 2)
unamplified cDNA reverse-transcribed from total mRNA, or 3) nuclear
genomic DNA (data not shown). This result argues that the second open
reading frame exists in the genome and that the sequence intervening
between the two open reading frames is not an unspliced intron. Second,
the length of the FP21 mRNA (800 bp) is not consistent with removal of
any conceptual intron in this region which can be constructed from
conventional Dictyostelium splice sites. Third, the amino acid
composition of purified FP21 is inconsistent with the composition for a
protein predicted to result if any conceptual intron is spliced out and
the resultant protein were to reflect the small open reading frame.
The most notable feature of the FP21 sequence is that it is devoid
of a signal peptide (33) for translocation across the rER or
any other sequences which are known to directly or indirectly promote
membrane translocation or anchorage. Based on comparison of predicted
and determined (5) NH termini, only up to two
NH
-terminal amino acids are removed from cellular FP21
after translation. These observations are consistent with the model in
which FP21 is both synthesized and glycosylated in the cytosolic
compartment, by glycosyltransferases which also reside in the cytosolic
compartment(5) .
No significant homology was detected with
other proteins in release 83.0 of the GenBank data base. The amino acid
sequence from 120-128 (Fig. 5) matches the ATP/GTP-binding
site motif A (P-loop) consensus sequence
((A/G)4GK(S/T))(34) . A sequence (amino acids
32-49, see Fig. 5) conforms to the prediction of the PEST
hypothesis for targeting rapid protein turnover(35) .
Significant
-helical folding is predicted for the first 40 amino
acids according to both Chou-Fasman and Garnier-Robson criteria, and
this region contains four heptad repeats which are predicted to fold
into a coiled coil domain (score = 1.33; (36) ) capable
of interacting with another polypeptide. This domain may explain the
aggregation characteristics of FP21 observed biochemically.
FP21, when tracked by radioactivity in its fucosyl moiety, was found previously to be highly enriched in the cytosolic fraction of lysed cells(5) . In this report, FP21's compartmentalization has been confirmed using antibodies, and new data about its carbohydrate composition and amino acid sequence provide additional strong evidence that this protein does not at any time enter the rER and is thus also glycosylated in the cytosolic compartment. The evidence can be summarized as follows.
First, when FP21 is tracked using specific antibodies, the majority of FP21 is found in the cytosolic fraction. This method of detection reveals, in addition, a substantial fraction of FP21 in the particulate fraction of the cell (Fig. 1). However, purified nuclei and purified actomyosin cytoskeletal preparations, isolated from cells extracted with different lysis mixtures, do not contain FP21, and membrane-disruptive agents do not release FP21 from the particulate fraction (Fig. 2). Rather, FP21 is released by high or low salt treatments (Fig. 2), which tend to dissociate protein-protein interactions. Consistent with this observation, soluble FP21 is capable of undergoing aggregation as determined by sedimentation studies (Fig. 4), SDS-PAGE, gel filtration high performance liquid chromatography (5) , and inaccessibility to in vitro fucosylation (Fig. 3). Possibly, FP21 is associated with a cytoskeletal fraction other than the actin-based system. Thus particulate FP21 appears to be in equilibrium with soluble FP21 in the cytosolic compartment.
Second,
the deduced amino acid sequence of FP21 (Fig. 5) predicts no
signal peptide which might promote translocation of FP21 into the rER
or other hydrophobic domains which might directly or indirectly promote
membrane association. No more than 2 NH-terminal amino
acids are normally cleaved after synthesis. The amino-terminal region
of the polypeptide is predicted to fold into a coiled coil domain,
which could support protein-protein interactions leading to
aggregation.
Third, monosaccharide composition analysis after acid
hydrolysis shows that FP21 does not contain GlcNAc or Man above
background levels and thus does not contain any N-linked
glycans. Since FP21 contains an N-glycosylation sequon at
amino acid residues 45-47 (Fig. 5) which might be utilized
if FP21 was exposed to the lumen of the rough endoplasmic reticulum,
the absence of N-glycosylation provides a functional
verification of the structural evidence that this protein does not
visit the lumen of the rER. FP21 glycosylation utilizes a pathway
distinct from that using the previously characterized cytosolic O-GlcNAc transferase (10) , based on the absence of
GlcNH after acid hydrolysis. The FP21 glycan is also
significantly different from an O-linked oligosaccharide on
the cytosolic enzyme phosphoglucomutase(4) , which is present
in Dictyostelium(
)and in other organisms. M
determinations (Table 1) indicate that
additional unknown posttranslational modifications occur on FP21. Since
the M
difference between wild-type and mutant FP21
is not explained by the differences in sugar or amino acid composition,
it follows that the unknown additional posttranslational modifications
are distinct in the wild-type and mutant strains.
The monosaccharide
analyses were performed on 5-10 µg of FP21 adsorbed to PVDF
membrane, an amount comparable with that used for protein
microsequencing, even though FP21 contains only about 3% carbohydrate.
Of the commercial membranes tested, Immobilon(TM)-P contained the lowest background levels of Glc and Xyl, but even
this membrane contains sufficient endogenous Glc to account for the
level of Glc detected in FP21. Protein was determined for parallel
replicated adsorbed samples by amino acid analysis. Protein
determination measurements, and comparison of acetone-precipitated and
PVDF membrane-adsorbed samples, allowed us to conclude that the
methodology described by Weitzhandler et al.(21) can
determine accurately for glycoproteins the absolute level as well as
the relative levels of neutral sugars, as previously reported using gas
chromatographic methods(38) . However, modified elution
programs are required to distinguish Xyl and Man, which often coelute.
FP21 resolves into two fractions, peaks I (20-25% of total) and II (75-80%), during DEAE anion exchange chromatography. These peaks contain distinct glycoforms as determined by monosaccharide composition analysis. Peak II contains approximately 2 mol of Gal, 1 mol of Xyl, 1 mol of Fuc, and 0-1 mol of Glc/mol of polypeptide chain, whereas peak I contains 1 mol of Gal, 2 mol of Xyl, 1 mol of Fuc, and 0-3 mol of Glc. Previous data indicated the presence of an O-linked, fucosylated tetra- or pentasaccharide on total pooled FP21(5) , and thus all sugars may occur as a single glycan. If so, the composition results suggest the existence of alternative glycosylation pathways. The negative charge previously reported (5) on this oligosaccharide is not due to phosphate or sulfate substituents, since FP21 could not be metabolically labeled with precursors of these substituents. It is unlikely to be due to a uronic acid, since no sugars with retention times similar to glucuronic or galacturonic acid were detected. It is also unlikely to be due to sialic acid, since there are no confirmed reports of this sugar in Dictyostelium. In the absence of fucosylation as a result of mutation, a single extra Xyl is applied to FP21. This is reminiscent of glycosylation pathways in human cells, where specific sialylation and fucosylation steps are mutually exclusive modifications of a core oligosaccharide structure(37) .
A transferase activity capable of fucosylating FP21 was previously detected in the cytosol(5) . The antibody tracking and sequence data reported here for FP21 substantiate the cytosolic localization of this enzyme, and the sugar analysis data strongly suggest the existence, in addition, of at least one novel galactosyltransferase and one novel xylosyltransferase, each capable of modifying proteinaceous acceptor substrates in the cytosol of Dictyostelium. Further evidence for these enzyme activities is currently being sought.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18063[GenBank].