From the Department of Anatomy and Cell Biology,
McGill University, Montreal, Quebec H3A 2B2, Canada,
¶ GlaxoWellcome Research and Development, Stevenage SG1 2NY,
United Kingdom, and
Département de Pathologie et Biologie
Cellulaire, Université de Montréal, Montreal, Quebec H3T
1J4, Canada
Received for publication, July 12, 2000, and in revised form, September 18, 2000
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ABSTRACT |
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A mass spectrometric analysis of proteins
partitioning into Triton X-114 from purified hepatic Golgi apparatus
(84% purity by morphometry, 122-fold enrichment over the homogenate
for the Golgi marker galactosyl transferase) led to the
unambiguous identification of 81 proteins including a novel
Golgi-associated protein of 34 kDa (GPP34). The membrane protein
complement was resolved by SDS-polyacrylamide gel
electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass
spectrometry characterization by peptide mass fingerprinting, tandem
mass spectrometry to generate sequence tags, and Edman sequencing of
proteins. Major membrane proteins corresponded to known Golgi
residents, a Golgi lectin, anterograde cargo, and an abundance of
trafficking proteins including KDEL receptors, p24 family
members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor,
and two SCAMPs. Analytical fractionation and gold immunolabeling of
proteins in the purified Golgi fraction were used to assess the
intra-Golgi and total cellular distribution of GPP34, two SNAREs,
SCAMPs, and the trafficking proteins GBF1, BAP31, and
The molecular mechanisms by which newly synthesized secretory
cargo is transported across the Golgi complex have remained elusive
since the discovery of the organelle (1). A cell-free transport assay
designed to reconstitute transport of secretory cargo through the
organelle has been instrumental in uncovering a dynamic coat complex
(via ARF/COPI coatomer) (2) and a membrane fusion machinery (via
NSF/SNAPs/SNAREs) (3). These constituents have been proposed to
regulate cargo selection, membrane budding, vesicular formation, and
membrane fusion to effect the anterograde delivery of newly synthesized
secretory proteins between adjacent Golgi-flattened cisternae (4).
Although recent studies have challenged this view (5-7), the relevance
of the above molecular machinery to the regulation of membrane
trafficking in the early secretory pathway remains unquestioned.
Parallel progress in understanding membrane traffic in the synaptic
terminal has been largely a consequence of a systematic analysis of the
major proteins identified therein largely as a consequence of the
cloning of their cDNAs. This has led to considerable insight into
the mechanisms of membrane targeting, fusion, budding, and vesiculation
at the synaptic terminal with several of the same proteins that were
identified via the Golgi cell-free transport assay (8-10).
Recent innovations in the use of mass spectrometry to characterize
proteins enable a rapid assignment of major proteins to cellular
structures, bypassing the requirement for protein characterization via
cDNA cloning. Hence, mass spectrometry-based protein assignments to
centrosomes (11) and the nuclear pore (12) have led to new views on the
function of these structures.
Edman degradation has been used previously to identify low molecular
weight integral membrane proteins of a highly purified hepatic Golgi
fraction. This led to the uncovering of four distinct members of the
p24 family of integral membrane proteins located largely in the
cis Golgi network and probably forming an intermolecular complex (13).
This approach has now been extended to the use of mass spectrometry
complemented by Edman degradation to identify all bands
visualized by one-dimensional
SDS-PAGE1 of Golgi proteins
partitioning into Triton X-114 and therefore expected to be membrane
proteins (14). The approach unambiguously identified 81 proteins. A
combination of electron microscope (EM) immunolabeling and
analytical centrifugation was used to visualize the subcellular
distribution of selected proteins identified by mass spectrometry and
Edman sequencing. Most abundant were Golgi resident enzymes and
membrane trafficking proteins including Rabs and SNAREs. A novel
peripheral membrane protein of 34 kDa was uncovered by this approach
and localized to the isolated Golgi apparatus by cryoimmune labeling
and to whole cells by confocal immunofluorescence. By systematically
defining the protein composition or proteome of each organelle of the
eukaryotic cell, a functional protein map of the cell may be realized.
Isolation of Golgi Fractions--
The WNG fraction was
isolated exactly as described by Dominguez et al. (7) and
characterized for protein yield and galactosyl transferase
marker enzyme enrichment also as described by Dominguez et
al. (7). Galactosyl transferase relative specific activity was
122 ± 32-fold (n = 3) enrichment over the
homogenate. The protein in the fraction corresponded to 0.043 ± 0.018% of the original homogenate protein.
SDS-PAGE, Triton X-114 Partitioning, and N-Terminal Edman
Sequencing of Golgi Membrane Proteins--
Triton X-114 phase
partitioning of Golgi integral membrane proteins was by the method of
Bordier (14), as described by Dominguez et al. (13). For
N-terminal Edman sequencing, phase-partitioned Golgi integral membrane
proteins were separated by SDS-PAGE, electrotransferred onto
polyvinylidene difluoride, and stained with Coomassie Brilliant Blue
(15). Bands were excised from the polyvinylidene difluoride blot and
washed with 20% methanol prior to automated microsequencing (16) on an
ABI 476A protein sequencer (PE Biosystems), employing a blot cartridge
and standard pulsed liquid blot protocol (17). Data were collected and
analyzed both by ABI 610A software and by manually overlaying
successive traces, and the resulting sequences were searched against a
nonredundant protein data base using FASTA (18), blastp (19),
and ProteinProspector MS-Edman programs. Signal peptides and the
number of transmembrane domains (four different algorithms) were
predicted according to algorithms available through the ExPASy
Molecular Biology Server. The algorithms were SignalP, HMMTOP, SOSUI,
TMHMM, and TMpred.
In-gel Enzymatic Digestion--
Protein bands were excised from
a Coomassie Blue-stained gel and destained prior to digestion according
to the published procedure by Shevchenko et al. (20). The
destaining was achieved by rinsing the excised gel pieces in 50%
acetonitrile in water (2 × 100 µl). Each rinse involved briefly
vortexing the sample prior to shaking at 37 °C for 5 min before
pipetting off the acetonitrile and adding 100 mM ammonium
bicarbonate (50 µl). The sample was vortexed again and shaken at
37 °C for 15 min before the addition of acetonitrile (50 µl). The
completely destained gel was then subjected to dithiothreitol (1 mg/ml)
to reduce cystinyl residues and then to iodoacetamide (10 mg/ml) to
effect alkylation. These reagents were removed, and acetonitrile was
added to re-shrink the gel pieces. The acetonitrile was removed, and
gel pieces were dried in a vacuum centrifuge. The dried gel pieces were
re-swollen in digestion buffer (50 mM ammonium bicarbonate
(pH 8.5), 5 mM CaCl2) containing 125 ng/10 µl
trypsin (Roche Molecular Biochemicals, sequencing grade). Digestion was
allowed to proceed overnight at 37 °C.
Peptide Analysis by Delayed Extraction Matrix-assisted Laser
Desorption Ionization (MALDI) Mass Spectrometry (MS)--
The
supernatant liquid from the digest was sampled directly as described by
Jensen et al. (21). An aliquot (0.4 µl) of the supernatant
was added to an equal volume of 5% formic acid previously spotted onto
a stainless steel target precoated with matrix. The matrix solution was
prepared by mixing a saturated solution of cyano-4-hydroxycinnamic acid
in acetone in a 4:1 ratio with a solution of nitrocellulose at 10 mg/ml
in acetone/propan-2-ol (1:1, v/v). The target was allowed to air dry
before being washed with 1% aqueous trifluoroacetic acid (2 µl).
Excess wash solution was blown off, and the target was dried using
compressed air. MALDI mass spectra were obtained using a ToFSpec
instrument (Micromass, Manchester, UK) fitted with a 337-nm nitrogen
laser. Spectra were acquired using the instrument in reflectron mode
and calibrated using a standard peptide mixture.
Peptide Analysis by Nanoelectrospray Ionization MS--
In cases
where the identity of the proteins present could not be established by
MALDI analysis alone, the peptides were further analyzed by
nanoelectrospray ionization MS/MS. Here the peptides were extracted
from the gel pieces using 100 mM ammonium bicarbonate (2 × 50 µl) and acetonitrile (2 × 50 µl) followed by
5% formic acid in 50% methanol (2 × 50 µl). The samples were
then centrifuged before removing the liquid into labeled tubes. The
combined extracts for each sample were then dried using a vacuum
centrifuge. Dried protein digests were redissolved in 5% formic acid
containing 5% methanol (10 µl) and desalted using a pulled-out glass
capillary containing a small amount of POROS R2 resin (PerSeptive
Biosystems). The desalting columns were prepared by slurry-packing a
minute amount of resin into the capillary. The columns were
equilibrated using 5% formic acid in 5% methanol (2 × 5 µl).
After loading the sample, the columns were washed using 5% formic acid
in 5% methanol (2 × 5 µl). The peptides were then eluted
directly into the nanospray needle using 5% formic acid in 50%
methanol (5 × 0.25 µl). The amount of packing material required
for this application is far less than that contained in commercially
available pre-packed columns, allowing the columns to be employed as
single use disposable items and avoiding the risk of sample loss and
contamination between samples. A Q-TofTM hybrid mass
spectrometer (Micromass) fitted with a Z-spray source was used
to acquire the mass spectra. In MS/MS mode the quadrupole is used to
select the precursor ion, which is then passed into a collision cell
where fragmentation is induced by collision with argon gas molecules.
The energy of collision is typically between 30 and 60 eV, depending on
the mass and charge of the precursor ion. Ions formed by the cleavage
of backbone bonds are designated a, b, c if the charge is retained on
the N-terminal fragment and x, y, z if the charge resides on the
C-terminal fragment (nomenclature according to Mann et al.
(22)). Product ions higher in m/z value than a
doubly or triply charged precursor ion are often part of a series of y"
ions (23). In many cases it is possible to read part of the sequence
from the pattern of y" ions. This, together with the masses bracketing
the sequence, forms a Peptide Sequence Tag (24) that can be used to
identify the peptide. Ions in the low mass region of the MS/MS spectrum
typically include b series ions as well as other internal fragments.
The latter are less specific but characteristic of the sequence
(25).
Antibodies to GPP34--
Rabbit anti-peptide antibodies were
raised against a peptide sequence (LKDREGYTSFWNDC; see Fig.
3B) derived from the human GPP34 sequence. The peptide was
coupled via the C-terminal cysteine residue to keyhole limpet
hemacyanin using
m-maleimidobenzoyl-N-hydroxysuccinimide ester as
the cross-linker. An initial subcutaneous injection of an emulsion of
peptide in complete Freund's adjuvant was followed by four boosts of
peptide (100 mg each) in incomplete adjuvant. Total IgG was purified by
protein A-Sepharose affinity chromatography (26). Rabbit antiserum was
also raised to a bacterial recombinant GST chimera of the human GPP34.
Polymerase chain reaction products encoding GPP34 cDNA (amplified
from IMAGE clone 664740, GB number AA232616, Research Genetics) were
purified by agarose gel electrophoresis, ligated into pGEX2T (Amersham
Pharmacia Biotech) and pTrcHisA (Invitrogen) using
EcoRI/BamHI as restriction sites, and transformed into Escherichia coli DH 5 EM Immunolocalization and Morphometry--
Freshly prepared
Golgi fractions were incubated with primary antibodies and processed
for EM immunolocalization as described by Lavoie et al.
(27). In the case of GPP34, cryosections of Golgi fractions were
prepared using the protocol description in Lavoie et al.
(27) and incubated with the anti-peptide antibodies to GPP34.
Confocal Laser Scanning Microscopy of Anti-GPP34--
GPP34
antigenicity was visualized in rat FR3T3 fibroblasts and in primary
cultures of rat hippocampal neurons. For neurons, the CA3 region of
PO-P1 rat hippocampi was dissected out, and the neurons were cultured
for 8 days as described in Baranes et al. (28). For
immunocytochemistry, neurons were fixed for 10 min with 4%
paraformaldehyde, 4% sucrose and incubated with affinity-purified rabbit antibody to recombinant GPP-34 (1:10) and mouse anti-MG160 (1:100; a kind gift of Drs. A. Beaudet, McGill University, and N. Gonatas, University of Pennsylvania). Neurons were then labeled with
goat anti-rabbit Cy3-conjugated (1:300) and goat anti-mouse fluorescein
isothiocyanate-conjugated (1:200) secondary antibodies (Jackson
ImmunoResearch Laboratories Inc.). Optical sections were obtained using
an LSM 410 confocal microscope (Zeiss), and images were taken under
nonsaturating conditions.
Analytical Fractionation of Total Membranes--
Livers from
rats fasted overnight were minced and homogenized in 0.25 M
sucrose, 4 mM imidazole buffer (pH 7.4) (homogenization buffer) with a motor-driven Potter-Elvejhem homogenizer with five strokes. The homogenate was filtered through two layers of cheese cloth, and the volume was adjusted (with homogenization buffer) to 20%
(w/v) of the starting liver wet weight. The homogenate was centrifuged
at 700 × gmax for 10 min. The resulting
supernatant was centrifuged at 200,000 × gmax for 40 min. The pellet (combined Mitochondrial, Light mitochondrial,
Particulate fraction (MLP)) was resuspended in 0.25 M sucrose imidazole buffer to 1 g/ml original liver weight,
and 0.5 ml of this sample was layered on top of a continuous 0.5-2.3
M sucrose gradient buffered in imidazole (pH 7.4) (total
volume 12.2 ml) and centrifuged at 110,000 × gmax for 17 h. Sixteen fractions were
collected from the top, with the weight and density measured in an Abbe
Mark II refractometer. Galactosyl transferase was assayed using
[3H]UDP-galactose and ovomucoid as substrate, as
described previously (29). Protein was estimated by the Bradford method
(30). Proteins of each fraction were precipitated with trichloroacetic
acid, washed with 70% ethanol, and then resuspended in 5 mM Tris-HCl (pH 8) with the aid of a sonicator on ice in
preparation for SDS-PAGE. Equal volumes of Laemmli sample preparation
buffer (31) were added, and the sample was heated in a boiling water
bath for 5 min. The resulting solution was clarified by centrifugation
in a microcentrifuge. For Western blotting, proteins in each
fraction were separated on a 10% gel by SDS-PAGE, and transferred to
nitrocellulose electrophoretically for 75 min at 12 V, employing a
Genie electrotransfer unit (IDEA Scientific Company, Minneapolis, MN).
The blots were blocked with 5% skim milk in 10 mM Tris,
150 mM NaCl, 0.5% Tween buffer (pH 7.5) (32) and then
incubated at 4 °C overnight with primary antibodies: 1:1000 for
protein A-purified anti-GPP34; 1:500 antibody dilution for SCAMP1 and
SCAMP3 (kind gifts of Dr. D. Castle, University of Virginia); 1:1000
dilution for
For some experiments the peroxidase chemiluminescent system (34), as
described by PerkinElmer Life Sciences, was used for visualization and quantitation. These experiments included GPP34 for Fig. 7A and mannosidase II, Identification of Integral Membrane Proteins by Gas Phase
Sequencing and Mass Spectrometry--
The Golgi fraction was selected
on the basis of its prior characterization, including a high enrichment
in the marker enzyme, galactosyl transferase, and diminution in
endosomal contamination (7). The WNG fraction was also characterized by
morphometry. Using a random sampling methodology (35), electron
microscopy of the filtered Golgi fractions revealed, as deduced from
the point hit method (36), that 84% of the profiles were Golgi
complexes. The remaining profiles were endoplasmic reticulum or plasma
membrane (14%), mitochondria (1%), or peroxisomal cores (1%), as
based on the analysis of 100 micrographs (× 27,000 final
magnification) and a point hit methodology employing 42 points per grid
(per micrograph).
N-terminal Edman sequencing (Fig. 1,
Table I) of integral membrane
proteins that partitioned into Triton
X-114 identified 18 proteins unambiguously, of which 11 were clearly
Golgi resident or trafficking membrane proteins. Readily identifiable
were the ER to Golgi trafficking proteins p58 (ERGIC 53), four members of the p24 family, two ERD2-like proteins (i.e. Elps or KDEL
receptors), mannosidase II, a GalNAc transferase, a sialyl transferase,
and a nonconventional GST (37). Three cargo proteins were found (paraxonase, apoE, and apoC III). Two putative endosomal proteins (p76
and EMP70) and two novel proteins (25 DX and GS3786) were found.
In contrast to N-terminal Edman sequencing, mass spectrometry
unambiguously identified 72 proteins (Fig. 1, Table I). As shown in
Table I, these were categorized into Golgi resident proteins,
trafficking proteins, contaminant (microsomal, mitochondrial, peroxisomal, endosome, or plasmalemma) proteins, and cargo proteins. No
proteins were deleted from the reported data either for analysis by
Edman sequencing or mass spectrometry.
Data analysis was effected as exemplified for band 16 (see Fig.
2, A-C, and Table II) of the
gel used for MS studies (Fig. 1,
right side). The major
component of this band was the Rab6 protein, based on the tryptic
peptide mass map. The ProteinProspector MS-Fit search algorithm for the
m/z values of the masses of the peptide fragments
confirmed that the masses shown in Fig. 2A indeed corresponded to Rab6, as deduced from the 26 peptide masses listed in
Table II. As seen in Fig. 2B, these peptides identified in the complete sequence covered 75% of the total sequence. However, only
the human Rab6 complete sequence is in the data base, whereas the
starting material is from rat liver. Only 26 of the 85 peptide masses
identified for band 16 corresponded to human Rab6. Removing those 26 and searching the data base with lower stringency, 6 more peptides were
assigned to Rab6, and peptide masses unique to Rab1a were
identified. Some peptide masses for Rab1a coincided with masses
of peptides for Rab6, confirming the similarity of predicted tryptic
peptide fragments. MS/MS sequence tag data (Fig. 2C) for the
doubly charged precursor at m/z 566.7, which
relates to a peptide of mono-isotopic molecular mass 1131.4 Da
((M + H)+ 1132.631, Fig. 2A) confirmed sequence
variation between the rat Rab6 protein and the DNA-predicted human Rab6
(Fig. 2B) and identified post-translational modifications of
the mature rat Rab6 protein.
Fig. 2C shows the rat Rab6 N-terminal sequence, identifying
that the initiation methionine was removed and that the protein is N-terminally acetylated. This is the first evidence for
N-acetylation of Rab proteins, although the consensus motifs for
removal of the N-terminal methionine and N-acetylation at the Ser
residue for Rab6 has been predicted (38). Screening the mouse EST data base (data not shown) confirmed the substitution of Ala at position 2 of the mature Rab6 protein for mouse as compared with the human sequence (Fig. 2, B and C). For all other
proteins identified by tryptic peptide mass mapping (Fig. 1, Table I),
total coverage was between 15 and 60%.
GPP34--
One protein of previously unknown function was
identified as a sequence conserved from yeast to humans (Fig.
3). One sequence tag (Fig. 3A)
identified a tryptic fragment (shown in italics in Fig. 3B)
that was found in the EST data base. Further searches led to the
alignment of the sequences shown in Fig. 3B. These corresponded to two human gene products, two mouse gene
products, and single gene products in Drosophila
melanogaster, Caenorhabditis elegans, and
Saccharomyces cerevisiae. Not shown are partial sequences matched to the data base of Schizosaccharomyces pombe,
Kluveromyces lactis, Aspergillus nidulans,
Danio rerio, and Xenopus laevis. Even though the
sequence has not been cDNA-cloned in any known species, the
corresponding gene (YDR372c) (Yeast Proteome Data Base) (39)
has been deleted from S. cerevisiae as part of the Saccharomyces Deletion Project (40). The gene is not
essential for viability, with no effect from gene deletion on growth
(Saccharomyces Deletion Project strain reference
numbers: 4208, 14208, 24208, 34208) (40).
The absence of a signal sequence and transmembrane domain predicts that
GPP34 is a peripheral membrane protein. Other peripheral proteins
partitioning into the membrane phase were GM130, p115, actin, myosin,
and ankyrin. GM130 and p115 are clearly Golgi-located (41),
cytoplasmically oriented, and bound to GRASP65 or GRASP55. GRASP65 and
GRASP55 are cytosolic proteins postulated to link adjacent
Golgi cisternae (42, 43) that were not detected in our analysis. The
presence of ankyrin in the fraction is expected (44), but its
partitioning into Triton X-114 implies tight association with an
integral membrane protein. In a similar way, the detection of myosin
and actin may be relevant to Golgi function. An unconventional GST with
three predicted transmembrane domains was identified (Table I). This
microsomal class of GSTs is unrelated by sequence to the conventional
GSTs, as deduced by blastp analysis.
Golgi resident proteins (Table I) including the Golgi marker MG160 (of
as yet unknown Golgi function but postulated to be a fibroblast growth
factor receptor (45)) and the processing enzyme mannosidase II,
also frequently utilized as a Golgi marker, were prominent. Other
N- and O-linked glycosyl processing enzymes were
found as expected, as was the Golgi- or cis Golgi-located lectin VIP36
(46, 47).
Trafficking proteins were identified. A 200-kDa protein with a sec7
domain was identified. This protein is the rat orthologue of a guanine
nucleotide exchange factor (GEF) for ARFs termed GBF1 and related to
products of the yeast genes GEA1 and GEA2 encoding Sec7 domains (48-50). The Sec7 domain of this class of proteins effects GDP-GTP exchange and when associated with ARF-GDP can
be targeted by the drug brefeldin A (48). However, the sequence-related protein p200ARF-GEF previously identified (51) and recently renamed
BIG1 (52) was not identified. In addition, only three Golgi v-SNAREs
(GS15, GS28, and Sec22b) were found. The Rab family GTPases were also
observed, of which Rab6 has been clearly Golgi-localized (53, 54)
whereas Rab5 is endosomal (55). Rabs 1a, 1b, and 2 (56-60) are
involved in ER to Golgi transport, and Rab7 is late-endosomal (61).
Rab8b is involved in TGN trafficking events (62). Rab10 has
been considered Golgi-located (63), and Rab13 may be in the plasma
membrane (64). In addition, p76 and the protein designated "similar
to EMP70" were found. An N-terminal fragment of the EMP70 protein was
originally identified in endosome fractions isolated from yeast and
called p24a (65). Two different gene products that have been identified
as homologues to yeast EMP70 were found in our study, i.e.
p76 (66) and the protein designated in the data base "similar to
EMP70" (67). Sequences corresponding to p76 and "similar to
EMP70" were observed by Edman degradation at 50, 147, and 165 kDa.
Remarkably, one of the two KDEL receptors (Elp-1b, Table I) was also
found at 147 and 165 kDa (besides its monomeric mobility at about 24 kDa). Conceivably, p76, "similar to EMP70," and the KDEL receptor
may associate into SDS-resistant complexes.
Unexpectedly, SCAMPs 1 and 3 were observed, as was a membrane protein,
BAP31, previously implicated as a regulator of apoptosis and suggested
to cycle between the ER and Golgi apparatus (68, 69). Members of the
p24 family, as well as p58, were found. Mammalian constituents (Sec23A
and Sec13) of the COPII complex postulated to bind to p58 and
the p24 family (13) were also identified. Remarkably, Sec22b, a v-SNARE
regulating ER to Golgi secretory cargo transport, was found (70-72).
Also found were the NSF-associated protein
MS/MS data were obtained from 40 individual peptides from 10 different
bands that we were unable to assign to a data base entry (data not
shown). This unassigned data could represent as many as 40 novel
proteins, but it is more likely that several peptides originate from
the same proteins, and therefore the number of novel proteins is
probably less than 40. Nevertheless, this represents an unexpectedly
high number of novel proteins whose significance remains to be elucidated.
Localization of Selected Trafficking Proteins and GPP34--
To
assess whether selected trafficking proteins were indeed in Golgi
membranes or contaminants, an immunolocalization study was effected. We
elected to study the intra-Golgi and cellular distribution of selected
trafficking proteins and GPP34 by electron microscope immunolabeling of
the Golgi fraction in situ and by analytical fractionation
of total liver membranes, respectively. Neither approach has previously
been used to address the distribution of these proteins.
Antibodies were raised to GPP34. Western blotting confirmed that the
antigen was membrane-associated as well as cytosolic (data not shown).
Immunolabeling of cryosections of the WNG fraction with the
peptide-specific antibody to GPP34 revealed specific labeling at the
periphery of the Golgi stack. Antigenicity was found on the cis and
trans sides as well as at the lateral edges of stacked cisternae (Fig.
4, indicated by arrowheads).
Controls without primary antibody but with secondary antibody
conjugated to gold revealed no detectable gold labeling in the fraction
(data not shown). Furthermore, confocal laser scanning confocal
microscopy of rat hippocampal neurons (Fig.
5) revealed a juxtanuclear staining (Fig.
5A) as well as a more diffused cytosolic labeling. The
juxtanuclear staining corresponded to that of the Golgi marker MG160
(Fig. 5B), with partial overlap observed (Fig.
5C). Labeling with anti-GPP34 alone gives identical staining
to that seen in Fig. 5A (data not shown). Similar
observations were made with rat FR3T3 cells (data not shown). Hence
GPP34 is in part Golgi-localized.
Direct immunolabeling of whole fractions by the method of Lavoie
et al. (27) revealed that the Golgi SNAREs identified here, i.e. GS15 and GS28, were both Golgi-located but with
different distributions (Fig. 6). GS15
was associated with edges of distended cisternae, whereas GS28 was
primarily in Golgi-associated smooth membranes as well as in Golgi
cisternae and vesicles. SCAMP1 was found on small Golgi-associated
structures, whereas SCAMP3 was found in larger structures. Stacked
flattened cisternae showed only low labeling. The ARF-GEF identified
here (i.e. GBF1) was largely in smooth membranes clearly
associated with stacked Golgi cisternae as well as in larger
lipoprotein-filled structures. BAP31 was found on ER contaminants but
also on associated vesicles and flattened cisternae of Golgi stacks. As
controls, the p24 family member
To identify the steady state proportion of these trafficking proteins,
which codistributed with the Golgi markers galactosyl transferase or
mannosidase II, analytical fractionation was employed (Fig.
7, A and B). The
rationale of analytical fractionation is that the sedimentation
properties of marker proteins define compartmental boundaries, with
median densities used as an index of comparison (73). Here GPP34
revealed a median density of 1.096, slightly less than that of the
Golgi markers galactosyl transferase (median density 1.122) and
mannosidase II (median density 1.120) (Fig. 7). Most of BAP31 and GS28
was found at higher median densities than that of the Golgi marker
(galactosyl transferase or mannosidase II), although some GS28 was
deduced to be Golgi-associated, as was the The elucidation of the membrane protein complement of an
organelle, i.e. the Golgi complex, was attempted by
organelle isolation, phase partitioning to extract the integral
membrane proteins, and protein characterization from one-dimensional
SDS-PAGE by Edman degradation and mass spectrometry. Although
two-dimensional gels are the preferred approach for proteomics, there
remains an unresolved experimental difficulty in the solubility of
membrane proteins during isoelectric focusing (74).
In the present study, the identified proteins were further
characterized for their location by immunolabeling and analytical fractionation. Indeed, for the SCAMP proteins, the Golgi SNARE GS28, and the apoptosis-related protein BAP31, this study
represents their first characterization by these approaches.
The methodology identified 81 proteins. These represented only the most
abundant membrane proteins, and further refined analysis suggests a far
greater complement in this Golgi
fraction.2 Of the 81 proteins
characterized, 49 were considered as integral membrane proteins on the
basis of having one or more predicted transmembrane domains
(Table I). In addition to the transmembrane proteins are those proteins
predicted to have covalent lipid modification motifs for
insertion into the membrane by lipid tails (about 12). Of all proteins
identified, 45 were considered to be in whole or in part Golgi-located.
These included 17 resident membrane proteins and 28 trafficking
proteins. Contaminants mainly from the ER and mitochondria were readily
identifiable and represented 24 proteins. In addition, 40 sequence tags
representing between 10 and 40 different novel proteins were
elucidated. The single full-length novel protein GPP34 identified by
gene building of mammalian EST sequences (Fig. 3) revealed deduced
sequence information from human to yeast. Although the full-length
cDNA cloning of this gene has not been reported, the gene has
already been shown to be nonessential for viability of S. cerevisiae (40), which does not exclude a regulatory role
for GPP34 in Golgi trafficking.
GPP34 revealed no targeting motifs, which might predict a Golgi
localization. Comparison of its distribution between membranes and
cytosol revealed it to be membrane-associated as well as cytosolic, as
deduced from subcellular fractionation (data not shown). Cryosections were used to ensure maximum availability of potential antigenic epitopes to the applied antibody. By the criterion of localization of
antigenicity on cryosections of the Golgi fraction, GPP34 was deduced to be Golgi-localized. Furthermore, by confocal fluorescence microscopy GPP34 was found in hippocampal neurons and fibroblasts to be
concentrated in regions overlapping that of the Golgi marker MG160. The
EST-derived primary sequence revealed no potential signal sequence or
transmembrane domain. Hence, the protein is most likely synthesized on
cytoplasmic ribosomes. Analytical subcellular fractionation revealed a
distribution and median density to slightly lower densities
than that of the two Golgi markers employed (galactosyl transferase and
mannosidase II). Most recently, Lin et al. (5) have
identified a recycling pathway for Golgi enzymes from Golgi cisternae
to the p24-containing cis Golgi compartment. The higher density tail in
analytical gradients of galactosyl transferase and mannosidase II
( The choice of Golgi fraction analyzed, i.e. the WNG
fraction, was a consideration of its prior characterization (7). In our
study, cargo was not depleted from the Golgi apparatus by agents such
as cycloheximide, in order not to compromise the distribution of any
membrane protein whose Golgi location may depend on cargo. Indeed,
continued protein synthesis is required for the localization of
membrane proteins such as BAP31 in post-ER compartments (68). Independently of its effect on protein synthesis, cycloheximide also
affects the secretory pathway directly, and this could also affect the
protein distribution in the Golgi (75). The problem of increased cargo
in the fraction was readily accommodated because few were expected to
be recovered in the detergent phase following extraction with Triton
X-114. A total of 10 candidate cargo proteins were identified by Edman
degradation or mass spectrometry, all of which were either amphipathic
or integral membrane proteins. However, whether endosomal proteins,
i.e. p76 and "similar to Emp70," are cargo or
contaminants requires further analysis.
It is noteworthy that by N-terminal Edman degradation, a different
cohort of integral membrane proteins was identified from that found by
mass spectrometry. N-terminal Edman degradation was superior for
identifying multipass integral membrane proteins. Hence, the two KDEL
receptors (i.e. Elp-1a and Elp-1b) and the two EMP70
homologues were identified by N-terminal sequencing but not by mass
spectrometry. Multiple transmembrane-containing proteins, such as
Elp-1a, Elp-1b, p76, and "similar to EMP70," may not be efficiently
in-gel-digested, and/or the tryptic peptides may not be efficiently
extracted after digestion for mass spectrometry. Indeed, none of the
tryptic fragments of any of the transmembrane proteins identified by
mass spectrometry (Fig. 1, right side) covered hydrophobic
transmembrane domains.
Implications for Membrane Trafficking Models--
The prominence
of SCAMPs was unexpected. Although they have been implicated in
endocytosis and exocytosis (76), no clear function is known for these
tetra span membrane proteins. The localization of SCAMP1 to Golgi
components predicts a role in membrane trafficking. Indeed, this is the
only speculative function for this family of membrane proteins,
i.e. in trans Golgi cycling compartments (76). Remarkably,
both SCAMPs showed a different intra-Golgi distribution by electron
microscopy (Fig. 6, C and D), whereas analytical
fractionation (Fig. 7A) revealed a similar distribution for
both and similar to that of the Golgi marker enzyme galactosyl transferase.
The Golgi complex is exquisitely sensitive to the drug brefeldin A,
with the action of this drug on Golgi structure and the inhibition of
secretion (77, 78) a consequence of the interaction of brefeldin A with
an ARF-GEF (50, 79). Two mammalian ARF-GEFs of 200 kDa with a Sec7
domain have been characterized to date (48, 49, 51). Only one of these,
GBF1 (48), was identified here as partitioning into Triton X-114.
Whether the other (BIG1) is also present requires further evaluation.
Interestingly, the majority of GBF1 was localized to elements apposed
to either side of the stacked Golgi cisternae (Fig. 6). Previous EM
studies have shown a cis Golgi location in flattened cisternae by
cryosectioning of intact liver (49). Efforts at more precisely
localizing GBF1 within the Golgi complex are underway. Also found in
domains apposed to one pole of the Golgi stack was 2P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although
GPP34 has never previously been identified as a protein, the
localization of GPP34 to the Golgi complex, the conservation of GPP34
from yeast to humans, and the cytosolically exposed location of
GPP34 predict a role for a novel coat protein in Golgi trafficking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Positive clones were
identified by polymerase chain reaction, and expression of the fusion
protein was confirmed by SDS-PAGE and Coomassie R-250 staining.
His-tagged GPP34 and GST-GPP34 were purified from
isopropyl-1-thio-
-D-galactopyranoside-induced bacterial
cultures and affinity-purified according to the manufacturer's instructions on NiNTA resin (Qiagen) or glutathione-Sepharose beads
(Amersham Pharmacia Biotech), respectively. Antibodies were raised as
above, employing complete Freund's adjuvant. Anti-GST-GPP34 antibodies
were further purified by affinity chromatography employing His-tagged
GPP34 bound to Sepharose 4B (Amersham Pharmacia Biotech), as described
by Harlow and Lane (26).
2p24, calnexin (CNX), and ribophorin II
rabbit polyclonal antibodies; 1:500 for the monoclonal antibody to
GS28; and 1:1000 for the chicken antibody to BAP31 (a kind gift of Dr.
G. C. Shore, McGill University). After each antibody reaction, the
blots were washed with 1% skim milk in 10 mM Tris, 150 mM NaCl, 0.5% Tween buffer (pH 7.5). In the case of GS28
monoclonal antibody or BAP31 chicken antibody, the washed blots were
incubated with rabbit anti-mouse or rabbit anti-chicken antibodies for
1 h at room temperature. For visualization and quantitation,
washed blots were incubated with a mixture of
125I-labeled goat anti-rabbit (2 µCi per blot) and
1:5000 dilution of goat anti-rabbit alkaline phosphatase, developed
with alkaline phosphatase reagent (32), and then exposed to x-ray film;
the respective bands were excised from each lane of the blot, and the
radioactivity was measured by a gamma counter. Distribution and
frequency were calculated as described in Dominguez et al. (13) using the methodology according to Beaufay et al.
(33).
2p24,
calnexin, and ribophorin II for Fig. 7B. For quantitation by
chemiluminescence, the Bio-Rad GS-710 densitometer linked to the
Multianalyst program was used. Protein A-peroxidase and goat anti-mouse
peroxidase were employed for detection of mannosidase II,
2p24, and CNX and ribophorin II, respectively, after
reaction with primary antibodies (1:1000).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
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Fig. 1.
Triton X-114 phase partitioning of the Golgi
fraction. N-terminal Edman sequencing and summary of mass spectral
characterization for identification of Golgi resident, trafficking,
contaminant, cargo, and novel proteins partitioning into the Triton
X-114 phase from the parent Golgi fraction. On the left are
indicated the proteins identified by N-terminal Edman degradation and
the assignment to bands from a Coomassie Blue-stained polyvinylidene
difluoride membrane blotted from an SDS-PAGE gel (10% acrylamide in
the resolving gel). On the right, integral membrane proteins
were electrophoresed in a 5-15% polyacrylamide gradient gel, and
Coomassie Blue-stained bands were characterized by mass spectrometry.
The numbers refer to band numbers identified as prominent
Coomassie Blue-stained polypeptides in separate gels (left,
Edman degradation; right, mass spectrometry). The mobilities
of molecular weight markers are indicated on the right of
the stained polyvinylidene difluoride membrane used for Edman
sequencing and on the left of the stained gel used for mass
spectrometry.
Characterization of WNG fraction proteins partitioning into Triton
X-114
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Fig. 2.
Characterization of Rab6 and Rab1a.
A, MALDI-time of flight mass spectrum for the tryptic digest
of WNG band 16 (Fig. 1, right side). Closed (see below) and
open circles identify Rab6 and Rab1a peptides, respectively. Internal
standards: cal-1 angiotensin (monoisotopic (M + H)+
1282.647) and cal2 ACTH 18-39 clip1 (monoisotopic (M + H)+
2465.162). The asterisk (m/z 1132.631)
refers to the peptide analyzed in C below. B,
coverage of the human Rab6 sequence (bold) based on the 26 matched tryptic peptide masses (Table II). AA's,
amino acids. C, nanoelectrospray ionization MS/MS of the
doubly charged (m/z 566.7) 1131.4-Da tryptic
peptide reveals this sequence tag fragmentation pattern, which
identifies the N-terminal tryptic fragment of rat Rab6. This peptide,
indicated by an asterisk in A ((M + H)+ 1132.6), was not assigned by MALDI peptide
mass mapping because of post-translational modifications (removal of
the initiation Met, acetylation of the N-terminal Ser (S-acet)) and
rodent-specific sequence variation of Ala for Thr at position 3 of the
predicted human sequence. The experimentally determined sequence of the
N-terminal tryptic peptide of mature rat Rab6 is acetyl-SAGGDFGNPLR.
This alanine to threonine sequence difference in the N-terminal tryptic
peptide (compare human Rab6, B) for rodent Rab6 was
confirmed in nine mouse ESTs (NCBI nr.05.07.99).
Summary of delayed extraction MALDI-MS data for band 16 (Fig. 1,
right-hand side) identifying Rab6
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[in a new window]
Fig. 3.
GPP34, a novel sequence identified by MS.
A, MS/MS spectrum of the doubly charged precursor at
m/z 772.0, which relates to a peptide of
monoisotopic molecular mass 1542 Da identified from a tryptic
fragment of band 24 (Fig. 1, right side). The data define
the sequence tag of (1270)PTGDV(801). B, alignment of the
GPP34. The sequence tag (1270)PTGDV(801) identified the translated
tryptic peptide SDAPTGDVLLDEALK (shown in italics for HGPP34
amino acids 134-149) that was confirmed by a complete y ion series in
the MS/MS spectrum in A (not indicated). Extension of this
sequence by searching the EST data base revealed the alignments
indicated in B. The alignment has been shaded by similarity
based on a PAM matrix. Degrees of shading indicate the levels
of similarity seen across the protein multiple sequence alignment
generated using the Matrix function of JavaShade (80) at a threshold of
1.0. Shown are human (H) sequences, mouse (M)
sequences, sequences of related (R) gene products, and the
deduced sequences of GPP34 in D. melanogaster
(GenBankTM accession number AC004340), C. elegans (GenBankTM accession number AC024791), and the
budding yeast S. cerevisiae (Yeast Proteome Database (YPDTM)
gene name YDR372C). The GenBankTM accession
numbers for the two human gene products HGPP34 and HGPP34R are AJ296152
and AJ296153, respectively.
SNAP and two TGN
trafficking proteins, i.e. the cation-dependent
mannose 6-phosphate receptor as well as TGN38.
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Fig. 4.
Localization of GPP34 in the Golgi
fraction. Cryosections of the Golgi fraction were labeled with
antipeptide antibodies to GPP34. Gold particle (10 nm) labeling is seen
at the periphery of stacked Golgi flattened cisternae on both cis and
trans sides. Arrowheads indicate gold particles;
Gc, Golgi cisternae. Bar, 100 nm.
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Fig. 5.
Colocalization of GPP34 and MG160 in
hippocampal neurons in culture. Primary cultures of hippocampal
neurons were double labeled for anti-GPP34 (Cy3, A) using
affinity-purified antibodies raised to recombinant GPP34 as described
under "Experimental Procedures" and anti-MG160 (fluorescein
isothiocyanate, B; same field as A) and imaged
with a confocal microscope. Merge (C) of A and
B. Scale, 20 µm.
2p24, which is
terminally N-glycosylated in this fraction, was cis located
as predicted from previous studies (13), and calnexin was primarily
found in ER contaminants (Fig. 6).
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Fig. 6.
Visualization of SNAREs, SCAMPs, trafficking
proteins, and ER contaminants. EM immunolocalization of GS15
(A), GS28 (B), SCAMP1 (C), SCAMP3
(D), GBF1 (E), BAP31 (F),
2p24 (G), and CNX (H).
Arrowheads identify gold particles (10 nm) indicating the
sites of antigenicity. All primary antibodies were to cytosolically
oriented epitopes of the respective proteins. Clumps of gold particles
considered an artifact of the secondary antibody are enclosed by a
circle in D as well as in F (far
right). The cis trans orientation of the stacked Golgi
cisternae is shown in F and G; mitochondria
(mit) are indicated in E. Smooth membranes
(SM) are indicated in G. ER and rough ER
(rER) are indicated in F and H,
respectively. The arrow in H identifies ribosomes
on a rough ER contaminant. The bars correspond to 500 nm.
2 member of
the p24 family. SCAMP 1 revealed a distribution similar to the Golgi
marker, as did SCAMP3. No GBF1 signal was detected, presumably because
of its dissociation from the MLP fraction as a consequence of the
preparation method and the imidazole buffer that was used (data not
shown). The GS15 signal was beyond detection in the MLP fraction by
Western blotting, presumably because the antibody is inefficient at
recognizing denatured protein in Western blots. We have, however,
previously detected GS15 by a chemiluminescence method of detection of
Western blots, but only in the purified Golgi (WNG) fraction (7) and
not in the total membrane fraction used for analytical
fractionation.
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Fig. 7.
Distribution of SNAREs, SCAMPs, trafficking
proteins, ER, and Golgi markers by analytical fractionation. Two
separate experiments (A and B) are illustrated in
which a combined total membrane preparation from rat liver homogenates
(MLP fraction) was fractionated by isopycnic sucrose gradient
centrifugation. In experiment A, the distribution of total
protein is compared with that of the Golgi marker enzyme galactosyl
transferase (GalT), the novel Golgi protein GPP34, SCAMP3,
SCAMP1, the p24 family member 2p24, the Golgi SNARE
GS28, the ER marker CNX, and the rough ER marker ribophorin II. The
median densities are indicated as vertical arrows. In
B is shown a second experiment in which the distribution of
mannosidase II is compared with that of
2p24, BAP31,
CNX, and ribophorin II. The distribution of
2p24
revealed a variation in experiment A compared with
B, whereas other control markers, i.e. ribophorin
II, calnexin, and the Golgi markers (galactosyl transferase
(A), mannosidase II (B)), reveal similar median
densities.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.12-1.19) could well correspond to a compartment harboring
recycling Golgi proteins, i.e. the cis Golgi network. The
peripheral membrane protein GPP34 appears to be absent from this latter
compartment. GPP34 antigenicity at the top of the gradient may
represent protein that dissociated from Golgi membranes during
centrifugation. These explanations may account for the slightly lower
median density of GPP34 than that of the Golgi markers. Two other novel
proteins (i.e. 25DX and GS3786) were identified by Edman
degradation. These integral membrane proteins have both been
cDNA-cloned; their function remains largely unknown, and their
sequences appear to be restricted to mammals. No information on their
cellular localization is known.
2p24,
a membrane protein previously identified in the cis Golgi network and
involved in ER to Golgi recycling mechanisms and in COPI
binding (5, 13, 27). The limited number of SNAREs, single ARF-GEF
partitioning into Triton X-114, and recycling membrane proteins from
the Golgi to ER (two Elps, p58, p24 family members) as prominent
constituents are unexpected. Indeed, the coincident distribuition of
the apoptosis-related protein BAP31 (69) with the cis Golgi marker
2p24 implicates the former in an ER to Golgi recycling
pathway. Characterization of the full-length sequences corresponding to
the 40 sequence tags with no corresponding sequences yet found in
current data bases may extend considerably the molecular machineries
regulating the function of the Golgi complex in
vivo.
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ACKNOWLEDGEMENTS |
---|
Valuable reagents were provided by Dr. A. Beaudet (McGill University), Dr. D. A. Castle (University of Virginia), Dr. G. C. Shore (McGill University), Dr. P. Melançon (University of Alberta), and Dr. Wanjin Hong (University of Singapore). Dr. P. Melançon also critically reviewed the manuscript before submission. We thank Line Roy for participation in several of the experiments in this paper.
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FOOTNOTES |
---|
* This work was supported by a Medical Research Council of Canada/Pharmaceutical Manufacturers Association of Canada grant (to J. J. M. B. and D. Y. T.) with GlaxoWellcome, Stevenage, UK and an MRC Genomics grant (to J. J. M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ296152 and AJ296153.
§ Both authors contributed equally to this work and should be considered co-first authors.
** To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, McGill University, 3640 University St., Montreal, Quebec H3A 2B2, Canada. Fax: 514-398-5047; E-Mail: bergeron@med.mcgill.ca.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M006143200
2 W. P. Blackstock and J. J. M. Bergeron, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SDS-PAGE, SDS-polyacrylamide gel electrophoresis; EM, electron microscope; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; GST, glutathione S-transferase; MLP, combined mitochondrial, light mitochondrial, particulate fraction; CNX, calnexin; ER, endoplasmic reticulum; EST, expressed sequence tag; GEF, guanine nucleotide exchange factor.
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