From the Genetic Disease Research Branch, NHGRI, National Institutes of Health, Bethesda, Maryland 20892
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The cytoplasmic face of the Golgi contains
a variety of proteins with coiled-coil domains. We identified one such
protein in a yeast two-hybrid screen, using as bait the peripheral
Golgi phosphatidylinositol(4,5)P2 5-phosphatase OCRL1
that is implicated in a human disease, the oculocerebrorenal syndrome.
The ~2.8-kilobase mRNA is ubiquitously expressed and abundant in
testis; it encodes a 731-amino acid protein with a predicted mass of 83 kDa. Antibodies against the sequence detect a novel ~84-kDa Golgi
protein we termed golgin-84. Golgin-84 is an integral membrane protein
with a single transmembrane domain close to its C terminus. In
vitro, the protein inserts post-translationally into microsomal
membranes with an N-cytoplasmic and C-lumen orientation. Cross-linking
indicates that golgin-84 forms dimers, consistent with the prediction
of an ~400-residue dimerizing coiled-coil domain in its N terminus. The dimerization potential is supported by a data base search that
showed that the N-terminal 497 residues of golgin-84 contain a
coiled-coil domain that when fused to the RET tyrosine kinase domain
had the ability to activate it, forming the RET-II
oncogene. Data base searching also indicates golgin-84 is similar in
structure and sequence to giantin, a membrane protein that tethers
coatamer complex I vesicles to the Golgi.
The Golgi cisternae constitute a compartmentalized and polarized
structure, allowing post-translational processing enzymes to act in
succession. As a routing center, the Golgi receives proteins from the
endoplasmic reticulum and routes them in bulk to the plasma membrane or
specifically to the endosomes and lysosomes. In polarized cells,
additional transport routes include targeting to the apical or
basolateral plasma membranes or to storage granules (1, 2). Protein
sublocalization, however, is not precise, since the locations of many
of the proteins overlap over two or more cisternae (3). All known
resident Golgi proteins are peripheral or integral membrane proteins.
Coiled-coil-containing proteins with different functions (4) have been
identified on the cytoplasmic face of the Golgi. C-terminally anchored
membrane proteins, with cytoplasmic coiled-coil domains, bind transport
vesicles to the cisternae membranes (5). One type tethers the vesicles
to membranes, while another type, termed SNARES, ensure that specific
vesicles dock to the membranes. Peripheral coiled-coil proteins on the
Golgi surface are of the resident and transient types; they maintain
Golgi structure and participate in budding, transport, tethering, and
docking of vesicles. Additional coiled-coil proteins of unknown
function have been identified as antigens in autoimmune diseases (6)
and include golgins 95, 160, and 245. One autoantigen, giantin-376,
(macrogolgin) has recently been shown to tether
COPI1 vesicles to the Golgi,
thereby facilitating vesicle transport within the Golgi stack (7). Here
we report the cloning and characterization of another, novel
cytoplasmic-associated coiled-coil integral membrane Golgi protein
termed golgin-84.
Yeast Two-hybrid Screen--
The yeast two-hybrid screen was
performed on a human brain cDNA library
(CLONTECH) using a MatchMaker kit
(CLONTECH) and a ~4.3-kb human OCRL1
cDNA (full open reading frame) as bait. The human OCRL1
cDNA was subcloned into vector pGBT9 (GAL4 DNA binding domain) and
the brain cDNA library into vector pGAD424 (GAL4 activating domain). The integrity of the OCRL1 construct was verified by in
vitro transcription and translation. Approximately 1.5 million brain clones were screened. Screening and elimination of false positives were performed as described by the manufacturer.
Screening Human Testis cDNA Library--
Plaque screening in
a Sequencing--
The brain and testis golgin-84 cDNAs were
cycle-sequenced (ABI 373A) as described by the manufacturer. Subclones
3, 12, and 16 were sequenced with universal primers FWD and REV and an
internal golgin-84-specific primer (13dS1). All three testis subclones were identical. Six additional golgin-84 primers (13dS2-13dS7) were
used to sequence clone 16: 13dS1 5'-GGTCATGCAGATCCTGTAAC-3', 13dS2
5'-TCAGCAGCTGATAACATTCG-3', 13dS3 5'-GCTGACCAGCTACTGAGTAC-3', 13dS4
5'-CATGAACTGTCTAACCTTCG-3', 13dS5 5'-GGCCAGATACATCAGCTCAG-3', 13dS6
5'-TCAACAGAACAATCATGACC-3', and 13dS7 5'-CGGAGTCCTCGAGTCATTCG-3'.
Sequence Analysis--
Protein and cDNA sequences were
analyzed by Lasergene (Macintosh) and PCGene
(IBM).2
Northern Blot Analysis--
A mouse multiple tissue Northern
blot (CLONTECH) containing 2 µg of
poly(A)+ RNA/lane was probed with radiolabeled 2.6-kb human
golgin-84 cDNA (specific activity, ~5.7 × 108
cpm/µg). The blot was prehybridized with ExpressHybTM
hybridization solution (CLONTECH) for 1 h at
68 °C and then hybridized in it for 2 h at 68 °C. After
hybridization, two 15-min room temperature washes of 2× SSC, 0.05%
SDS were performed followed by two 30-min washes at 60 °C. The blot
was then exposed for 4 days. After exposure, the blot was stripped,
prehybridized, and hybridized with a Antibody Production--
Antibodies were raised in rabbit
against denatured and solid phase renatured His6 tag
protein containing golgin-84 amino acids 499-687. A 500-ml E. coli culture containing the golgin-84 pET construct (Novagen) was
induced at A595 ~0.5 with 2 mM
isopropyl
Antisera were raised in an accelerated program; three rabbits were each
immunized at different sites with denatured and solid phase renatured
golgin-84 (Zymed Laboratories Inc.). Antibodies were
purified on a golgin-84 affinity column prepared with CNBr-Sepharose 4B
(Amersham Pharmacia Biotech). The purified golgin-84 antibodies were
eluted from the column with 3 M KSCN, pH 7.0, and dialyzed at 4 °C overnight against 10 mM Tris, pH 7.0.
Protein Electrophoresis and Immunoblotting--
Protein
concentrations were determined with a Bio-Rad protein assay kit II.
Cell and tissue lysates (30 µg/lane) were separated on 6-12%
SDS-polyacrylamide gels and then electroblotted (110 V-h) onto
polyvinylidene difluoride (Millipore Corp.) membrane. The integrity of
the Western blots was analyzed by Coomassie Blue staining. Destained
membranes were blocked with 3% gelatin (1 h) and then incubated with
golgin-84 primary antibody (1:1000) in PBS containing 1% gelatin (room
temperature for 1 h). Immunostained proteins were detected with an
horseradish peroxidase-labeled secondary antibody (Amersham Pharmacia
Biotech) and enhanced chemiluminescence (ECL) reagents (Amersham
Pharmacia Biotech).
Light Microscopy--
For light microscopy, cells were grown in
four- or eight-chambered slides (Lab-Tek) using standard tissue culture
techniques. Whole mouse testes were prepared by rapid freezing in OCT
compound (Miles) followed by cryosectioning (10 µm) and air drying
the sections on slides. Cells and sections were fixed with 4%
paraformaldehyde for 10 min. (room temperature) before permeabilization
with either 0.2% Triton X-100 for 15 min or with 0.2% saponin (Sigma)
added to all solutions.
Fixed samples were immunolabeled with the affinity-purified polyclonal
golgin-84 antibody (1:1000) as well as with commercial monoclonal
antibodies mannosidase II (clone 53FC3, Babco), protein-disulfide isomerase (clone RL90, ABR) and
For fluorochrome labeling, cells were fixed, blocked with 10% serum,
and then probed with primary and secondary antibodies in PBS.
Fluorescein isothiocyanate- and Texas Red-labeled secondary antibodies
were used at their recommended working dilutions (Jackson Laboratories). After immunostaining, cells were PBS-washed and mounted
with Immunofluor mounting media (ICN).
Horseradish peroxidase immunostaining was accomplished with
Vectastain® Elite ABC (peroxidase) and VECTOR® VIP substrate kits (Vector Laboratories). The protocols were carried out essentially as
described in the kits with the exception that additional blocking was
performed with avidin and biotin (Vector Laboratories). Immunostained testis sections were also counterstained with methyl green (Vector Laboratories) prior to mounting with Permount®.
Immunostained samples were observed by fluorescence epiillumination and
bright field microscopy on a Leica DMRBE microscope. Images were
recorded on Kodak Ektachrome Slides (400 ASA) and then scanned and
processed in Adobe Photoshop 4.
In Vitro Transcription and Translation--
Golgin-84 number 16 cDNA was subcloned into Bluescript II SK+ with its 5'-end oriented
toward the T7 promoter. The construct DNA was digested with
SpeI and phenol chloroform-extracted. Golgin-84 mRNA was
then transcribed from the T7 promoter and capped using an mRNA
capping kit (Stratagene). Capped golgin-84 mRNA was translated (0.2 µg/25 µl) for 2 h at 30 °C in a rabbit reticulocyte lysate (Promega) containing [35S]methionine (Amersham Pharmacia
Biotech) label and canine pancreatic microsomes (Promega). After
translation, the microsomes were collected by centrifugation (10 min,
12,000 × g), washed three times with PBS, and
incubated in 0.1 M Na2CO3, pH 11.0, at 4 °C for 30 min to remove peripheral membrane proteins. The
stripped microsomes were centrifuged at 4 °C and resuspended in 1× PBS.
In the proteinase protection study, CaCl2 and Tris (pH 8.0)
were added to the translation mix and put on ice for 15 min. before the
proteinase K was added. The final concentrations were 10 mM CaCl2, 10 mM Tris (pH 8.0), and 0.2 µg/µl
proteinase K. Proteinase K digestion was performed for 1 h at
4 °C, after which the microsomes were pelleted (4 °C, 10 min) and
resuspended in a protease stop buffer (15 µl of PBS, 5 µl of 0.1 M phenylmethylsulfonyl fluoride, 5 µl of 5× sample
buffer), and the resuspended microsomes were immediately boiled for 5 min. As a positive translocation control,
To analyze the mechanism of golgin-84 insertion into microsomal
membranes, translation was terminated with a final concentration of 25 µg/µl cycloheximide. Microsomes were added to the terminated translation reactions and incubated for 2 h at 30 °C before
being PBS-washed and alkaline carbonate-stripped.
In cross-linking studies, PBS-washed (two times) golgin-84-containing
microsomes were exposed to 0.01 and 0.10 mM
dithiobis(sulfosuccinimidylpropionate) (DTSSP) in PBS (1.8 µl of
Promega microsome stock/50 µl of DTSSP) for 30 min at room
temperature. The cross-linking reactions were quenched with 50 mM Tris, pH 7.5 (final concentration). Cross-linked protein
was analyzed by SDS-PAGE under nonreducing conditions, since the DTSSP
cross-linker is thiol-cleavable.
After separation by SDS-PAGE, the in vitro translated
products were electroblotted to polyvinylidene difluoride membrane and sprayed with En3Hance spray (DuPont) before being exposed to Kodak X-Omat AR film.
Enhanced Green Fluorescent Protein (EGFP)-tagged Golgin-84
Deletion Constructs, Plasmid Construction, and
Expression--
EGFP-tagged fusion proteins were expressed in cultured
cells from cytomegalovirus promoters. Golgin-84 cDNA restriction
fragments encoding the C-terminal 51 (BclI, XbaI)
and 184 (BglII, XbaI) amino acid residues were
subcloned into BglII, XbaI-digested pEGFP-C3 DNA
(CLONTECH). Expression constructs were made with
nonmethylated DNA and screened for in a methylase minus E. coli strain SCS110 (Stratagene). Once made, the constructs were
transformed into E. coli DH5a. DNA was then prepared for
transfection using Qiagen kits. Twenty-four hours prior to
transfection, normal rat kidney (NRK) cells were seeded (~1.6 × 105 cells/ml) into chambered (four or eight chambers)
slides (Lab-Tek) coated with poly-L-lysine (Sigma). Cells
were transfected by exposing them to serum-free DMEM containing DNA and
liposomes (1 µg of DNA/5 µl of LipofectAMINE (Life Technologies,
Inc.) in 1 ml of medium), and after 5 h an equal volume of medium
containing 20% fetal bovine serum was added. Transfected cells were
analyzed by light microscopy ~20 h after the start of transfection.
For immunostaining, cells were fixed for 10 min with 4%
paraformaldehyde and permeabilized with 0.2% saponin. A pEGFP vector
without insert was expressed in cells as a control.
Yeast Two-hybrid Screen
An adult human brain cDNA library was screened with a 4.3-kbp
human OCRL1 cDNA (8) using a Matchmaker kit. Of ~1.5
million clones screened, two positives were identified. One clone was a
novel 781-bp partial cDNA termed 13d. Based on its size and subcellular localization (see below) we term the 13d protein golgin-84. A search of the Unigene data base indicated that the golgin-84 gene
maps to distal human chromosome 14q in the region D14S292-qter.
Golgin-84
mRNA Is Ubiquitously Expressed and Abundant in Testis--
To
analyze golgin-84 expression a mouse multiple tissue poly(A)+ mRNA
blot was probed with a golgin-84 cDNA (Fig.
1). A single ~2.9-kb message was
detected that was ubiquitously expressed and abundant in testis.
Similar results were obtained with human poly(A)+ mRNA
blots (message ~2.8 kb, data not shown). A 2.63-kbp golgin-84 cDNA (number 16) was then isolated from a human testis library with
a 828-bp expressed sequence tag probe, 134426 (Fig.
2). The testis clone was found to lack
~0.2 kbp of sequence from its 3'-UTR when compared with expressed
sequence tag AA405331, which matched 85 bp at the 3'-end of clone 16 and extended an additional 209 bp and included a poly(A)+
tail. The G + C content was uniform across most of the cDNA but was
increased significantly (68%) in the 5'-UTR (131 nucleotides). This
high G + C content is characteristic of housekeeping promoters and
5'-UTRs. Of all of the ATGs analyzed in the golgin-84 number 16 cDNA, the sequence flanking the 5'-most ATG (CCATCATGT)
conformed best with the Kozak consensus (9, 10) as determined by a weighted matrix. An in frame stop codon occurred 21 nucleotides upstream of the first ATG. The open reading frame extending from this
ATG was predicted by the Fickett algorithm (11) to have a 92% chance
of coding and encoded a 731-amino acid protein with a mass of 83 kDa.
In vitro transcription and translation of the golgin-84
cDNA yielded an ~84-kDa protein, which matches the protein size
seen by Western blotting of tissues (described below). We conclude
therefore, that the combined sequence contains the entire coding region
and most if not all of the golgin-84 transcript.
A Ubiquitous 84-kDa Protein Abundant in Testis--
To study the
golgin-84 protein, antibodies were raised in rabbit against
His6-tagged denatured and solid phase renatured antigen containing golgin-84 amino acids 498-688 made in E. coli.
Golgin-84 antibodies were purified from rabbit antisera with an
affinity column. One affinity-purified antibody immunoprecipitated
in vitro translated 35S-labeled golgin-84
protein and detected an ~84-kDa protein on Western blots of human
fibroblast lysates. The corresponding preimmune serum did neither,
implying that the affinity-purified antibody recognizes both native and
denatured golgin-84.
Golgin-84 protein expression was next analyzed in tissues. The
affinity-purified golgin-84 antibody was used to probe a Western blot
containing lysates (30 µg/lane) from eight mouse tissues, the same
tissues used for Northern blot analysis (Fig.
3A). Golgin-84 protein was
abundant in testis and present at much lower levels in the other
tissues, consistent with the Northern blot data. In heart and muscle,
the antibody cross-reacted with abundant myosin (data not shown).
To study golgin-84 expression further, mouse testis cryosections were
histochemically immunostained with the affinity-purified golgin-84
antibody. Immunostained sections indicated that golgin-84 was abundant
in the seminiferous tubules and Leydig cells (Fig. 3B). Not
all seminiferous cells immunostained with the antibody; staining was
greatly reduced or absent in the spermatozoa. The immunostaining
pattern differed between seminiferous tubules, probably reflecting
differences in the stage of spermatogenesis occurring in different
tubules (12). The juxtanuclear immunostaining pattern and its
absence/reduction from mature spermatozoa suggested that golgin-84
localized to the Golgi.
A Resident Golgi Protein--
To determine if golgin-84 was
sublocalized to the Golgi, NRK cells were double-labeled with the
affinity-purified golgin-84 antibody and an antibody against the Golgi
enzyme mannosidase II (medial-trans cisternae). Golgin-84 was detected
by fluorescein isothiocyanate and mannosidase II with Texas Red-labeled
secondary antibodies. The fluorescent signals from the two proteins
overlapped considerably, indicating that golgin-84 is indeed a Golgi
protein (Fig. 4A). Consistent
with golgin-84 being a Golgi protein, anti-golgin-84 antibody labeled
fragmented structures in mitotically dividing NRK cells (13); the
mitotic status of the cells was established by double labeling with a
To establish the resident time of golgin-84 in the Golgi, NRK cells
were treated with 50 µg of cycloheximide/µl of culture media for 1, 2, 4, and 7 h. Cycloheximide inhibits protein synthesis without
affecting protein traffic across the Golgi compartments, thus clearing
the Golgi of proteins in transit. At 7 h, golgin-84 was still in
the Golgi, implying that it is resident there (data not shown).
BFA-induced Redistribution Kinetics Is Similar to Known Golgi
Integral Membrane Proteins--
To confirm golgin-84's
sublocalization as well as to examine its redistribution kinetics on
Golgi perturbation, NRK cells were exposed to 10 µg/ml brefeldin A
(BFA) in tissue culture media (14). After a 1-h incubation with BFA,
golgin-84 was transported to and distributed throughout the endoplasmic
reticulum (Fig. 4B). Several cells still showed an intense
staining immediately adjacent to their nuclei, suggesting that
golgin-84 may be present in the trans-Golgi in addition to the earlier
disassembling cis/middle cisternae (15). Shorter BFA exposure times of
2, 5, 15, and 30 min showed that golgin-84 began to redistribute to the
endoplasmic reticulum (ER) at between ~2 and 5 min of BFA treatment
(data not shown), similar to integral membrane Golgi proteins such as mannosidase II (data not shown), but much slower than rapid on-off proteins involved in coat formation such as A C-terminally Anchored Type II Integral Membrane Protein That
Post-translationally Inserts into Membranes--
Golgin-84 was
predicted to be a transmembrane protein with type II topology.
Hydropathy profile analysis (18) predicted a single transmembrane
domain (TMD) close to its C terminus at amino acid residues 699-717
(Fig. 5A) with a slight
hydrophobic moment on an Eisenberg hydrophobic moment plot (19). The
hydrophobic moment suggests the golgin-84 TMD associates with one or
more other TMDs in the membrane (19). Based on the charge flanking the
golgin-84 TMD, the N-terminal 698 residues of the protein were
predicted to be cytoplasmic, and the short 14-residue C terminus was
predicted to be luminal (type II membrane protein). No signal sequence
was detected with a von Heijne weighted matrix algorithm (20),
suggesting that the protein inserts post-translationally into
membranes.
To determine if golgin-84 is an integral membrane protein, its mRNA
was translated into [35S]methionine-labeled protein in
the presence of canine pancreatic microsomes. After translation, the
microsomes were washed, stripped to remove peripheral membrane
proteins, and collected by centrifugation. The microsomes contained
golgin-84, indicating that the protein had been translocated into them
(Fig. 5B).
The membrane location and orientation of golgin-84 was then established
by a protease protection assay. Microsomes containing the translocated
protein were treated with proteinase K. Golgin-84 was digested by the
protease to below 66.2 kDa, suggesting that the 698-amino acid N
terminus was exposed on the surface of the microsomes, consistent with
it being a type II membrane protein (Fig. 5, C and
D). A control secretory protein
Golgin-84 lacks a predicted signal sequence and is thus expected to
undergo post-translational insertion into membranes (21). To test this
hypothesis, microsomes were added either prior to the start of
translation or after translation had been terminated by the addition of
cycloheximide (Fig. 5E). Microsomes that were exposed to
translating or full-length golgin-84 protein (translation terminated by
cycloheximide) for a period of 2 h at 30 °C were washed with
PBS, alkaline-extracted, and analyzed by SDS-PAGE. As expected,
translating golgin-84 was translocated into the microsomes. Full-length
golgin-84 protein, exposed to microsomes after termination of
translation, was also translocated into the microsomal membranes, implying a post-translational mechanism of insertion.
The C-terminal 51 Residues Contain the Membrane Insertion Sequence
and Golgi Retention Signal--
In vitro transcription and
translation studies indicated golgin-84 to be a C-terminally anchored
membrane protein. To confirm that the protein is C-terminally anchored
as well as to determine if its C terminus contains membrane insertion
and Golgi retention sequences, the C-terminal 51-amino acid region of
golgin-84 was tagged at its N terminus with EGFP and expressed in
cultured NRK cells (Fig. 6).
EGFP, when expressed by itself, was present throughout the cells. In
contrast, the tagged 51-residue C terminus (fusion protein I) was
targeted to the Golgi. In low expressing cells, the fusion protein (I)
was predominantly in the Golgi, while in high expressing cells it
accumulated in the ER as well. The ER and Golgi were distinguished by
their morphology as well as by immunostaining with antibodies against
mannosidase II (Golgi) and protein-disulfide isomerase (ER). Once in
the Golgi, fusion protein I was effectively retained there, and no
fluorescent signal could be detected on the plasma membrane. The
presence of fusion protein I in the Golgi and its absence from the
plasma membrane indicate that the C-terminal 51 residues of golgin-84
contain membrane insertion and Golgi retention sequences. In addition,
the absence of coiled-coil sequence in the C terminus implies that
coiled-coil interactions are not required for Golgi retention, as
suggested by an oligomerization-based model of Golgi retention (3).
Finally, backup of fusion protein I in the ER of high expressing cells
suggests that (a) the protein inserts into the ER membrane
and is then transported to the Golgi, as has been reported for giantin,
a Golgi protein of similar structure (22) and (b) export
from the ER is inefficient because the fusion protein (I) lacks an ER
export signal (23, 24) or is detained by the ER quality control system
(25, 26).
Since the 51-residue C terminus was inefficiently transported to the
Golgi, a larger tagged construct encoding the last 184 residues of
golgin-84 (fusion protein II) was made and expressed in NRK cells (Fig.
6). The larger fusion protein was transported efficiently to the Golgi
in both low and high expressing cells. Endogenous golgin-84 could not
be co-immunoprecipitated with fusion protein II, suggesting that it did
not interact with the fusion protein and is not involved in
transporting it to the Golgi (data not shown). A putative ER export
signal present in fusion protein II but absent in I may be responsible
for its more efficient transport to the Golgi. The sequence of amino
acid residues DTE, located 37 residues N-terminal of the golgin-84 TMD,
may ensure efficient recruitment of the protein into COPII vesicles, a
requirement for transport to the Golgi (23, 24). Overexpression of
fusion protein II in COS-7 cells caused Golgi fragmentation with no
plasma membrane labeling, indicating that the Golgi retention mechanism was not saturated and therefore not receptor-dependent
(data not shown).
Protein Dimerization--
Golgin-84 contains a putative
~400-amino acid coiled-coil domain in its cytoplasmic N terminus. Two
leucine zippers were identified in the golgin-84 sequence (residues
227-248 and 301-322) by a prosite pattern search. Since leucine
zippers are a specialized type of coiled-coil, a coils program (version
2.1) was used to analyze the extent of the coiled-coil domain (27, 28).
The coiled-coil domain, containing discontinuities within it, spans more than half the length of the protein; its boundaries were predicted
at amino acid residues ~217 and 632 using a window of 28 and an
arbitrary cut-off probability of >90%. The coiled-coil domain was
also visible as internal heptad repeats on a dot matrix plot of
golgin-84 against itself generated with a moving window of 30 and
cut-off of 35% (data not shown).
Golgin-84 was predicted by a multicoil program (29) to dimerize via its
coiled-coil domain (Fig. 7A).
To investigate if golgin-84 dimerizes, PBS-washed microsomes containing
35S-labeled golgin-84 were incubated at room temperature in
0.01 and 0.10 mM DTSSP, a membrane-impermeable
cross-linker. As predicted, cross-linked golgin-84 migrated by SDS-PAGE
as dimers. The amount of cross-linked dimers increased with increasing
cross-linker concentrations. At 0.01 mM DTSSP, monomers and
dimers were present, while at 0.10 mM no monomers remained
(Fig. 7B).
Golgin-84 Is Similar to Coiled-coil-containing Golgi
Proteins--
To gain insight into golgin-84 function homology,
searches were performed against available World Wide Web data bases
such as GenBankTM. Golgin-84 shows significant sequence
similarity to several coiled-coil-containing proteins, including Golgi
proteins. The strongest similarity is to the RET-II oncogene
(BLAST p value = 6.2e
Golgin-84 shows sequence similarity to the coiled-coil-containing
myosin family (BLAST p value
<5.8e Golgin-84 is a novel C-terminally anchored Golgi protein with an
extensive cytoplasmic coiled-coil domain. Golgin-84 was established as
an integral membrane protein based on its hydropathy profile and its
inability to be extracted in vitro from microsomal membranes with a high pH buffer. Protease protection studies confirmed a predicted type II topology. The post-translational mode of golgin-84 insertion into membranes is consistent with its lack of a signal peptide and the presence of its TMD within the last 50 residues of the
protein (21, 36).
Golgi targeting sequence(s) were shown by EGFP fusion studies to be in
the last 51 residues of the golgin-84 protein, suggesting its
transmembrane domain may be involved in Golgi retention. A sequence
comparison of Golgi and plasma membrane TMDs indicates that Golgi TMDs
are on average 5 residues shorter than plasma membrane TMDs (37).
Consistent with this observation, synthetic TMDs 17 leucines in length
are retained in the Golgi, while those 23 leucines in length are
transported to the plasma membrane (38). The Golgin-84 TMD is predicted
to be 19 residues. A lipid-sorting model has been proposed to explain
TMD retention by length. The model suggests post-Golgi membranes are
thicker (sterols and sphingolipids), thereby preventing the shorter
Golgi TMDs from moving forward (38). Oligomerization (kin selection)
has also been suggested to play a role in the retention of some
resident Golgi proteins (39).
The golgin-84 coiled-coil domain spans more than half the length of the
protein, suggesting that the molecule is rod-like in shape.
Discontinuities within the coiled-coil domain may create fixed bends or
provide flexibility to the structure (40). The coiled-coil domain
further suggests that, like myosin II, golgin-84 could form dimers, and
we confirmed this by in vitro cross-linking studies.
Golgin-84 dimerization may explain its ability to constitutively activate the RET-II oncogene. Included in RET-II are the
first ~274 residues of the golgin-84 coiled-coil domain with its two leucine zippers, which may activate RET-II by constitutive
homodimerization. Constitutive leucine zipper-mediated homodimerization
has been reported to activate another RET oncogene termed
PTC1 in which a fragment of a coiled-coil protein H4 is
fused to the RET tyrosine kinase domain (41, 42). Unlike
PTC1, which is rearranged with RET in 11-25% of
papillary thyroid carcinomas, the RET-II rearrangement appears to be an artifact created during transfection of NIH3T3 cells
with human colon cancer DNA, since the rearrangement was not detected
in the original tumor DNA by Southern blot analysis (30).
The function of golgin-84 is unknown, but some clues are provided by
its ubiquitous expression, Golgi sublocalization, and structure and
sequence similarity to Golgi proteins of known function. Abundant
expression in the testis indicates that it may serve a specialized role
there such as in the formation of the acrosome. Alternatively, or in
addition, its abundant testis expression may reflect the secretory
potential of that organ.
Coiled-coil integral membrane proteins with structures similar to
golgin-84 have been implicated in specific docking or in tethering
(Velcro factor) of transport vesicles (5). Specific docking proteins
termed SNARES ensure that transport vesicles fuse with their
appropriate target membranes. A vesicle SNARE (v-SNARE) interacts with
a specific target SNARE (t-SNARE) on the acceptor membrane. At first
glance, golgin-84 does not appear to be a SNARE, since a prosite
profile search indicates it lacks the t-SNARE homology domain reported
previously (43). Golgin-84 does, however, show structural and sequence
similarity to a larger 350-kDa tethering protein giantin (34, 35). Like
golgin-84, giantin is a type II C-terminally anchored membrane protein
with an extensive cytoplasmic coiled-coil domain. Giantin also forms homodimers and lacks a t-SNARE domain. Giantin has been shown to tether
COPI vesicles to Golgi membranes, thereby facilitating protein
transport between Golgi cisternae (7). Being as much as four times the
width of COPI vesicles, giantin is thought to increase the efficiency
of intra-Golgi transport by ensuring COPI vesicles budding from one
cisterna become attached to the next before budding is complete. In the
tethering process, giantin becomes linked to a series of other
proteins; giantin first binds p115 (7), p115 in turn associates with
GM130 (44), and finally GM130 interacts with GRASP65 (45). The COPI
docking process is inhibited during mitosis by GM130 phosphorylation,
resulting in Golgi fragmentation. Golgi fragmentation during mitosis is essential to ensure the stochastic distribution of the organelle between daughter cells (13). Golgin-84, in view of its structure and
sequence similarity to giantin, may play a similar role tethering transport vesicles to target membranes. Consistent with this
speculation are electron micrographs of immunostained human fibroblasts
that show golgin-84 on vesicles (~80-nm diameter) in the perinuclear region. The identity of these vesicles remains to be
established.3
A role for giantin in maintaining Golgi structure has also been
proposed. Giantin may link adjacent cisternae within the Golgi stack
(35), or it may form a carpet of fibrous attachment proteins that guide
transport vesicles within the Golgi stack (46). It has been suggested
that giantin is attached to the Golgi cytoskeleton and plays a
structural role. Supporting a cytoskeleton association, giantin
localization is little affected when cells are extracted with 1%
Triton X-100 in a microtubule-stabilizing buffer prior to fixation; in
contrast, other Golgi membrane proteins such as galactosyltransferase
were completely extracted (35). A similar detergent extraction
experiment resulted in complete removal of golgin-84. In that
experiment, mannosidase II was also mostly extracted, while the
microtubule cytoskeleton, although slightly disrupted, remained
intact.3 Golgin-84, therefore, does not appear to associate
with the cytoskeleton.
Taken together, the existing data suggest that golgin-84 plays a role
in vesicle tethering/docking. Additional analysis will be needed to
confirm such a role as well as to verify any interaction the protein
has with OCRL1. If golgin-84 is proven to be involved in
tethering/docking, then it should lead to a better understanding of
Golgi-associated vesicle transport.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
lgt10 human testis 5'-Stretch Plus cDNA library
(CLONTECH) was performed using double lifts onto
nitrocellulose filters (DuPont) from 12 Petri dishes (150-mm diameter)
containing ~37,000 plaques on an NM514 Escherichia coli
lawn. A golgin-84 expressed sequence tag probe 134426 (828 bp) was
[32P]dCTP-labeled (Amersham Pharmacia Biotech) using a
Prime-ItTM RmT labeling kit (Stratagene) and
NucTrapTM probe purification columns (Stratagene). After a
4-h prehybridization, the filters were hybridized with the labeled
golgin-84 probe under high stringency (50% formamide, 6×
saline/sodium phosphate/EDTA, 5× Denhardt's solution, 1% SDS, 5%
dextran sulfate, 0.1 mg/ml sheared salmon sperm DNA) at 42 °C
overnight. Blots were washed twice at room temperature with 2× SSC,
0.1% SDS for 15 min each and then at 65 °C for the same time. The
blots were finally washed with 0.2× SSC and 0.1% SDS at 65 °C for
15-30 min. Seventeen plaques were positive through the tertiary
screen. Inserts were then amplified from the purified phage with
lgt10 arm primers (CLONTECH) and confirmed as
golgin-84. DNA was purified (Qiagen) from three phage (numbers 3, 12, and 16) with inserts closest in size to full-length golgin-84 mRNA
(~2.8 kb), and the inserts were subcloned into EcoRI-digested pUC18 (Amersham Pharmacia Biotech). The
largest subclone, number 16 (2.6 kbp), was missing ~200 bp from its
3'-end.
-actin probe (specific
activity, ~4.45 × 108 cpm/µg) as described for
golgin-84. Washing conditions were the same as for golgin-84, although
an additional high stringency wash of 0.1× SSC, 0.1% SDS was
performed at 60 °C for 1 h.
-D-thiogalactopyranoside (final concentration)
for ~4-5 h. Cells were harvested by centrifugation and lysed
overnight at 4 °C in 6 M guanidine hydrochloride, 50 mM NaH2PO4, pH 8.0, and the lysate was cleared by centrifugation at 8000 rpm. The cleared supernatant was
passed over a Ni2+ resin column (pretreated as described by
the manufacturer, Qiagen), and the column was then washed with ~25
bed volumes of lysis buffer. Protein was eluted from the column in a
denatured form by the addition of 6 M guanidine
hydrochloride, 50 mM NaH2PO4 in
aliquots of decreasing pH: pH 6.0 (2 × 5 ml), pH 5.0 (3 × 3 ml), and pH 3.5 (1 × 10 ml). The denatured protein was then
subjected to SDS-PAGE (nine gels, ~1 mg of antigen/gel), cut from the
gels, and used for immunization. Solid phase renatured protein was also
prepared on the column by a stepwise renaturation procedure; 25 bed
volumes of MCAC (20 mM Tris·Cl, pH 7.9, 0.5 M
NaCl, 10% glycerol, and 1 mM phenylmethylsulfonyl
fluoride)/6 M guanidine hydrochloride (v/v) buffers mixed
in ratios of 1:1, 3:1, and 7:1 were added to the column in the given
order. Each renaturation buffer contained 20 mM imidazole.
Solid phase renatured protein was eluted from the column with MCAC
buffers containing increasing concentrations of imidazole, in
increments of 20 mM. The NaCl concentration of the MCAC
buffer was lowered to 0.15 M (instead of 0.5 M)
immediately prior to and including the elution step in order to
facilitate the immunization procedure. Eluted protein was concentrated
by ultrafiltration in centricon filtration units (cut-off
Mr 3000). Eight milligrams of solid phase
renatured golgin-84 protein was prepared at a concentration of 1.5 mg/ml.
-tubulin (clone Z023,
Zymed Laboratories Inc.). The commercial antibodies
were used at dilutions recommended by their manufacturers. Primary
antibodies were detected with fluorochrome and horseradish
peroxidase-labeled secondary antibodies.
-lactamase was used to
demonstrate the microsome membranes were functional and intact.
RESULTS
View larger version (46K):
[in a new window]
Fig. 1.
Golgin-84 mRNA is ubiquitously expressed
and abundant in testis. A Northern blot containing mouse
poly(A)+ RNA (2 µg/lane) from eight different tissues was
probed with the 2.6-kb human golgin-84 cDNA. A human actin control
probe was used to compare mRNA levels between lanes. The murine
golgin-84 message is ~2.9 kb, ubiquitously expressed, and is abundant
in testis. There is no evidence of alternate splicing.
View larger version (91K):
[in a new window]
Fig. 2.
Human golgin-84 cDNA sequence and its
encoded protein (GenBankTM accession number AF085199).
The first 2.63 kb of sequence was derived from a testis cDNA; the
remaining 0.2 kb of 3'-UTR sequence, shown after the dashed
line, was taken from expressed sequence tag AA405331. The
largest open reading frame is predicted to have a 92% chance of coding
(11); it encodes a 731-amino acid protein of ~83 kDa. The sequence
flanking the ATG initiation codon conforms with the Kozak consensus and
is preceded by an in frame stop codon 21 nucleotides upstream. A
polyadenylation signal, AATAAA, is located immediately upstream of the
poly(A) tail.
View larger version (57K):
[in a new window]
Fig. 3.
Golgin-84 is a ubiquitous ~84-kDa
protein abundant in testis. A, an immunoblot of mouse
tissue lysates labeled with affinity-purified golgin-84 polyclonal
antibodies and detected by ECL. B, golgin-84 is abundantly
expressed in seminiferous tubules and Leydig cells of testis.
Immunostaining is restricted to a juxtanuclear region characteristic of
the Golgi. Mouse testis cryosections were immunostained with
affinity-purified golgin-84 antibodies and detected histochemically
with a Vectastain ABC kit and Vector VIP substrate. The tissue sections
were counterstained with methyl green (bar, 2.5 µm).
-tubulin antibody (data not shown).
View larger version (27K):
[in a new window]
Fig. 4.
Golgin-84 is sublocalized to the Golgi.
A, NRK cells were double-labeled with antibodies against
golgin-84 and a Golgi marker protein mannosidase II. Golgin-84 was
detected with fluorescein isothiocyanate-labeled secondary antibodies
and mannosidase II with Texas Red-labeled secondary antibodies
(bar, 2.5 µm). B, golgin-84 is redistributed to
the ER in BFA-treated NRK cells. NRK cells were exposed to 10 µg/ml
BFA in culture media for 1 h, after which time the BFA was removed
and the cells were allowed to recover for an additional 1 h. After
1 h of BFA treatment, golgin-84 had been reabsorbed into and
distributed throughout the endoplasmic reticulum. An intense
juxtanuclear signal was also present in some cells, indicating that
some of the protein may be associated with the trans-Golgi network. One
hour after the removal of BFA, golgin-84 had regained its Golgi type
distribution, although the recovery process was still incomplete
(bar, 2.5 µm).
-COP (16, 17). Upon drug
removal, the Golgi reformed, and golgin-84 returned to it. Cells
recovered at different rates. By 1 h, many cells appeared almost
fully recovered, with small amounts of golgin-84 remaining in the ER
(Fig. 4B).
View larger version (32K):
[in a new window]
Fig. 5.
Golgin-84 is a C-terminally anchored type II
integral membrane protein that inserts post-translationally into
membranes. A, golgin-84 was predicted to be an integral
membrane protein with a single transmembrane domain located close to
its C terminus (amino acids 700-717). The hydropathy profile was
plotted as the mean hydrophobic index against amino acid number, using
a moving window of 19 amino acids with a 1-residue interval (18).
B, golgin-84 was in vitro translated in the
presence of [35S]methionine and canine pancreatic
microsomes. After translation, the microsomes were washed twice with
PBS and extracted with an alkaline buffer to remove peripheral membrane
proteins. Golgin-84 was retained with the microsomes, suggesting that
it was translocated into them. C, the N terminus of
translocated golgin-84 is exposed on the surface of the microsome.
Microsome-translocated golgin-84 was digested by proteinase K to below
66.2 kDa, suggesting that the 700-amino acid N terminus was exposed to
protease. A control protein, -lactamase, was translocated and
protected, indicating that the microsomes were functional and intact.
D, schematic diagram of golgin-84 translocated into a
microsome. E, golgin-84 lacks a signal sequence and
undergoes post-translational insertion into membranes. When translated
in the presence of microsomes, golgin-84 undergoes translocation via
either a co- or post-translational route. Cycloheximide added before
translation (bfT) inhibits translation of golgin-84. The
addition of cycloheximide after translation (afT) but before
microsome addition resulted in translocation, indicating that the fully
translated golgin-84 protein is capable of inserting into membranes.
Computer analysis did not identify a golgin-84 signal sequence,
consistent with a post-translational mechanism of insertion.
-lactamase was translocated into the microsome lumen and protected from digestion, indicating that the microsomes were functional and intact (Fig. 5C).
View larger version (15K):
[in a new window]
Fig. 6.
The C-terminal 51 residues of golgin-84
contain membrane insertion and Golgi retention sequences.
A, schematic diagram of wild type and mutant EGFP-tagged
golgin-84 proteins. Fusion protein I contains the last 51 amino acid
residues of golgin-84, and fusion protein II contains the last 184. B, an EGFP tag expressed by itself appeared throughout NRK
cells. Fusion protein I in contrast was targeted to the Golgi
(mannosidase II immunostaining) and was not detected on the plasma
membrane, indicating that the C-terminal 51 residues of golgin-84
contain both membrane insertion and Golgi retention sequences. A
reticular and perinuclear staining pattern, also observed for fusion
protein I, suggested that the protein was present in the ER. The ER
localization was confirmed by immunostaining with a protein-disulfide
isomerase antibody (data not shown). The presence of fusion protein I
in the ER suggests that it inserts there but is inefficiently
transported to the Golgi. The larger fusion protein II was transported
efficiently to the Golgi, suggesting that an ER export signal occurs
between residues 547 and 680 of the golgin-84 protein. Consistent with
this suggestion, a DTE sequence was identified 37 residues N-terminal
of the golgin-84 TMD (bar, 2.5 µm).
View larger version (24K):
[in a new window]
Fig. 7.
Golgin-84 forms dimers. A,
golgin-84 is predicted to dimerize via its coiled-coil domain (29). B,
in the presence of DTSSP, golgin-84 is cross-linked to form dimers. The
membrane-impermeable cross-linker DTSSP was added at different
concentrations to microsomes containing 35S-labeled
golgin-84. Golgin-84 dimers are indicated by the
arrow.
305),
which it turns out consists of the first 497 amino acid residues of
golgin-84 fused to residues 713-1114 of the RET protein (30). RET
residues 713-1114 contain a tyrosine kinase domain that is activated
by dimerization (31, 32). Activation of the kinase domain in RET-II is
probably achieved by constitutive dimerization induced by the golgin-84
coiled-coil sequence. An earlier RET-II report presented the
partial golgin-84 protein sequence but neither characterized it nor
suggested a mechanism by which it activates RET-II (33).
16) and to several coiled-coil Golgi
proteins (range of BLAST p values was
2.2e
14 to 4.6e
9).
Golgin-84 is similar to six known Golgi proteins: giantin
(macrogolgin), golgin-160, Golgi complex autoantigen-97, golgin-245,
trans-Golgi p230, and cis-Golgi matrix protein GM130. Sequence
similarities are associated primarily with the ~400-residue
coiled-coil domain. The functions of most of these Golgi proteins are
currently unknown, many having been identified as autoantigens.
Golgin-84 is structurally similar to one of the autoantigens, known as
giantin, a C-terminally anchored integral membrane protein with an
extensive cytoplasmic coiled-coil domain (34, 35). The golgin-84
coiled-coil domain and its immediate flanking sequence (residues
173-642) are 45% similar (22% identical) to the giantin coiled-coil
sequence (residues 1322-1806). Recently, giantin was shown to tether
COPI vesicles to the Golgi and has been proposed to play a role in
stacking Golgi disks (7).
DISCUSSION
![]() |
FOOTNOTES |
---|
* 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) AF085199.
To whom correspondence should be addressed: GDRB/NHGRI, 49 Convent
Dr., Bethesda, MD 20892-4472; Tel.: 301-402-2039; Fax: 301-402-2170;
E-mail: rlnuss{at}nhgri.nih.gov.
The abbreviations used are: COPI and COPII, coatamer complex I and II, respectively; PBS, phosphate-buffered saline; DTSSP, dithiobis(sulfosuccinimidylproprionate); UTR, untranslated region of mRNA; NRK, normal rat kidney; BFA, brefeldin A; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein; TMD, transmembrane domain; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); kbp, kilobase pair(s); bp, base pair(s).
2 Additional analyses, including data base searching, were performed at various World Wide Web sites, listed at http://www-biol.univ-mrs.fr/english/logligne.html.
3 R. A. Bascom, S. Srinivasan, and R. L. Nussbaum, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|