From the Diabetes Branch, NIDDK, National Institutes
of Health, Bethesda, Maryland 20892 and the § Institute
for Genome Research, Rockville, Maryland 20850
Received for publication, May 30, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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Sorting nexins are a family of phox
homology domain containing proteins that are homologous to yeast
proteins involved in protein trafficking. We have identified a novel
342-amino acid residue sorting nexin, SNX15, and a 252-amino acid
splice variant, SNX15A. Unlike many sorting nexins, a SNX15 ortholog
has not been identified in yeast or Caenorhabditis elegans.
By Northern blot analysis, SNX15 mRNA is widely expressed. Although
predicted to be a soluble protein, both endogenous and overexpressed
SNX15 are found on membranes and in the cytosol. The phox homology
domain of SNX15 is required for its membrane association and for
association with the platelet-derived growth factor receptor. We did
not detect association of SNX15 with receptors for epidermal growth
factor or insulin. However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to
their mature subunits. Immunofluorescence studies showed that SNX15
overexpression resulted in mislocalization of furin, the endoprotease
responsible for cleavage of insulin and hepatocyte growth factor
receptors. Based on our data and the existing findings with yeast
orthologs of other sorting nexins, we propose that overexpression of
SNX15 disrupts the normal trafficking of proteins from the plasma
membrane to recycling endosomes or the trans-Golgi network.
Intracellular vesicle traffic in both yeast and mammals requires
the function of numerous proteins that mediate multiple processes including cargo selection, vesicle budding, and fusion of vesicles with
specific targets (1-3). Sorting nexins
(SNXs)1 are a family
of widely expressed proteins believed to be part of the complex
molecular machinery required for protein trafficking (4). Mammalian
SNXs are homologous to several yeast proteins (e.g. Vps5p,
Mvp1p, and Grd19p) for which there is strong genetic evidence
demonstrating a role in protein trafficking (5-7). SNX1 was identified
using the yeast two-hybrid system by virtue of its ability to bind to
the cytoplasmic domain of the epidermal growth factor (EGF) receptor
(8). Subsequently, SNX1 was shown to also interact with receptors for
insulin, platelet-derived growth factor (PDGF), transferrin, and leptin
(4). Furthermore, SNX1 is the mammalian ortholog of Vps5p, a yeast
protein that is a component of a multimeric complex (termed the
"retromer complex") involved in retrograde transport of proteins
from prevacuolar endosomes to the TGN (9). We have previously described
three additional sorting nexins: SNX2, SNX3, and SNX4 (4); and 10 additional sorting nexins (SNX5-14) have been deposited in
GenBankTM. Like SNX1, both SNX2 and SNX4 associate with
various receptors. In addition, SNX1, SNX2, and SNX4 assemble into
oligomeric structures (4). All 14 SNX molecules contain a phox homology
(PX) domain, a conserved sequence of unknown function first identified
in the p40phox and p47phox subunits of the
NADPH oxidase complex (10). PX domains consist of ~100 amino acid
residues, and most contain a proline-rich sequence that may represent
an SH3 domain-binding motif (10). PX domains have been identified in
>20 proteins, some of which are involved in protein trafficking in
yeast (6, 9). Using a PX domain consensus sequence obtained from
SNX1-4 to search the NCBI data base, we identified a cDNA encoding
a novel SNX protein, SNX15.
Cloning of SNX15 cDNA--
We determined a consensus
sequence for the phox homology domain of SNX1, 2, 3, and 4 (GenBankTM accession numbers U53225, NP0030901, NP003786,
and NP003785, respectively), using clustal W (11). We searched the NCBI
data base with this consensus sequence using the BLAST algorithm and identified seven human expressed sequence tags (ESTs) corresponding to
a novel sorting nexin, now designated SNX15. Five ESTs (accession numbers T65368, AA351071, F11999, R17390, and AA351142) were obtained
from Research Genetics (Huntsville, AL), and plasmid DNA was isolated
using reagents provided by CLONTECH (Palo Alto, CA). Using appropriate vector- and sequence-specific primers we determined the nucleotide sequences of the ESTs. The sequence data
revealed two groups of SNX15 EST clones. The first group of ESTs
encoded a long form of the molecule, designated SNX15. The second group
of ESTs encoded a presumed splice-variant of SNX15, designated SNX15A
that lacks 270 bp at the 3' end of the cDNA. Neither group
contained a 5' start site. To obtain additional 5' sequence including
the protein start site, we carried out PCR with a Tissue Distribution of SNX15 mRNA--
Multiple tissue
Northern blots containing ~2 µg of purified human
poly(A)+ RNA and normalized for equal actin loading were
obtained from CLONTECH. To obtain a probe for
Northern blot analysis, SNX15 cDNA was excised from the
epitope-tagged pcDNA3.1+ plasmid construct with XbaI and
NotI. The resulting fragment was gel purified using reagents
supplied by Qiagen (Chatsworth, CA), and labeled with 32P
using a random primed DNA labeling kit (Roche Molecular Biochemicals) and diluted in Express Hybridization Solution
(CLONTECH). Blots were hybridized at 60 °C for
16-18 h and washed according to the manufacturer's instructions.
Construction of Expression Vectors--
Using PCR catalyzed by
Klen-taq (CLONTECH), we introduced c-Myc and
influenza hemagglutinin (HA) epitope tags at the 5' end of SNX15 and a
c-Myc tag at the 3' end of SNX15A. In addition, using similar methods,
we constructed several mutants of SNX15. Antibodies--
Antibodies to the Transient Expression of Sorting Nexins and Receptors--
COS7
cells (American Type Culture Collection, Manassas, VA) were maintained
in Dulbecco's modified Eagles medium (DMEM), supplemented with 10%
(v/v) fetal bovine serum (Life Technologies, Inc.), 100 units/ml
penicillin, and 100 µg/ml streptomycin. 2-3 × 105
cells were plated into individual wells of a 6-well plate 18-20 h
prior to the start of each transfection. Cells were then washed twice
with phosphate-buffered saline (PBS). DNA for transfection was prepared
according to manufacturer's instructions (Life Technologies, Inc.).
DNA (0.8-1.0 µg total/well) in OPTI-MEM (100 µl/well) (Life Technologies, Inc.) was precomplexed with PLUS Reagent (6 µl/well) with later addition of LipofectAMINE (4 µl/well). Cells
were transfected for 3-5 h with the DNA-LipofectAMINE complexes in
serum free DMEM and then incubated overnight in 10% (v/v) fetal bovine
serum. The cells were harvested 24-26 h after the start of
transfection. For immunofluorescence studies, COS7 cells were seeded at
1 × 105 cells/side of a two-chambered slide (Nunc
Inc., Naperville, IL) 18 h before transfection. Transfections were
performed with LipofectAMINE 2000 (Life Technologies, Inc.) following
the manufacturer's instructions, using a total of 3 µg of plasmid
DNA/6.25 µl of LF2000 reagent/well, with an incubation time of
4 h.
Immunofluorescence--
Transfected cells were fixed in 2%
formalin (v/v) in PBS, incubated with the primary antibody in PBS, 10%
fetal bovine serum (v/v), and 0.075% saponin (w/v) for 1.5 h,
washed, and then incubated with labeled secondary antibody in the same
buffer for 45 min (20). Cells were then viewed in a Zeiss Axiophot
inverted microscope (Carl Zeiss Inc., Thornwood, NY). Images were
captured with a PentaMAX camera (Princeton Instruments Inc., Trenton,
NJ) and IP Labs software (Scanalytics Inc., Fairfax, VA). Adobe
Photoshop (Adobe Systems Inc., Mountain View, CA) was used to process
the images.
Coimmunoprecipitation Experiments--
Transfected COS7 cells
were washed in ice-cold PBS and scraped into 250 µl of cold lysis
buffer/well (50 mM Tris-HCl, pH 7.5, 0.5% (v/v) Triton
X-100, 0.3 M NaCl, Plus protease inhibitor tablet) (Roche
Molecular Biochemicals). The cells were solubilized on ice for 30 min
and centrifuged at 14,000 × g for 20 min at 4 °C to
remove insoluble debris. Proteins were detected by standard immunoblotting procedures or by immunoprecipation of extracts (400 µl) with specific antibodies (1:100 dilution; see "Antibodies"). Immune complexes were sedimented with 30 µl of Ultralink immobilized protein A or protein G (Pierce). The beads were washed and boiled in
Laemmli sample buffer. Proteins were separated by SDS-PAGE (7.5 or
12.5%). Proteins were then transferred to nitrocellulose (21), and
epitope-tagged proteins were detected by Western blot followed by
chemiluminescence using ECL reagents (Amersham Pharmacia Biotech).
Subcellular Distribution of SNX15--
COS7 cells (2-3 × 105 cells/well), transiently expressing Myc-tagged SNX15 or
mutant forms of SNX15 (see above), were placed on ice and washed twice
with ice-cold PBS. Cells were scraped and lysed in 250 µl of ice-cold
homogenization buffer (10 mM HEPES, pH 7.4, 0.25 M sucrose, 1 mM ethylenediaminotetraacetate,
0.5 mM MgCl2, and protease inhibitor tablet)
(Roche Molecular Biochemicals) by 15 passages through a 25-gauge
needle. To obtain total membrane and cytosolic fractions, the lysates
were centrifuged at 240,000 × g for 45 min at 4 °C
in a Beckman TLA120.2 rotor (Beckman Instruments, Palo Alto, CA).
Following centrifugation, the pellet was resuspended in 250 µl of
homogenization buffer using eight strokes with a Teflon pestle and a
Potter homogenizer. Aliquots of the total lysates, as well as cytosolic
and membrane fractions, were solubilized in Laemmli buffer and
separated on 12.5% (w/v) polyacrylamide-SDS gels. Epitope-tagged
molecules were detected by immunoblotting with anti-Myc antibody and
ECL reagents obtained from Amersham Pharmacia Biotech.
Metabolic Labeling Studies--
Labeling of COS7 cells
(2-3 × 105 cells/well) was preformed after 2 h
of preincubation in methionine- and cysteine-free DMEM containing 0.1%
(w/v) bovine serum albumin, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Cells were labeled for 20 min with 100 µCi/ml of
EasyTag Express Protein Labeling Mix (1175 Ci of 35S/mmol;
Amersham Pharmacia Biotech) in methionine- and cysteine-free DMEM (2 ml). Labeling was terminated by washing the cells twice with ice-cold
PBS and replacing with prewarmed (37 °C) DMEM containing 2 mM cysteine and methionine, 10% (v/v) fetal bovine serum,
100 units/ml penicillin, and 100 µg/ml streptomycin. After chasing for the times indicated in figure legends, media were removed, cells
were washed twice in ice-cold PBS and frozen on liquid nitrogen. Cells
were scraped, and lysates were prepared by solubilization in ice-cold
Kahane buffer + octylglucoside (20 mM octylglucoside, 0.5%
Triton X-100, 0.3 M NaCl, 0.025 M sodium
phosphate, pH 7.4, and 0.02% NaN3) plus Complete protease
inhibitor (Roche Molecular Biochemicals). Cell lysates were spun at
14,000 rpm for 20 min, and the supernatants were then precleared by
incubating with 30 µl of protein A-agarose (Pierce) for 3 h on a
rotating wheel at 4 °C. Samples were subsequently centrifuged
(14,000 rpm for 1 min), and then the supernatants were incubated
overnight at 4 °C with specific antibody (see figure legends).
Antibody complexes were sedimented using 30 µl of protein A-Sepharose
(Pierce) for 3 h at 4 °C on a rotating wheel. Protein A pellets
were washed twice with Kahane buffer + octylglucoside, once with Kahane
buffer, and once with Tris-buffered saline. Gel samples were prepared (see above), and the proteins were separated by SDS gel electrophoresis and transferred to nitrocellulose. The blots were directly exposed to
film and quantified by scanning densitometry using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
125I-PDGF Internalization and Degradation--
Cells
were transfected with an expression vector encoding the PDGF
Phylogenetic Analysis of the Sorting Nexins--
PX domain
sequences were identified by an eight-round PSI-BLAST (23) search with
the PX domain of SNX4 as the seed, and an initial stringency of
1e Identification and Tissue Distribution of SNX15--
In an effort
to identify additional members of the sorting nexin family, we compared
the predicted amino acid sequences of SNX1-4 (4). The PX domain was
the only region of homology shared by all four molecules. A consensus
sequence for the PX domain was created using the clustal W algorithm
(11) and then used to search the dbEST data base of the NCBI. We
identified multiple ESTs encoding portions of a new sorting nexin,
SNX15. We determined the complete sequence of two ESTs, both of which
lacked the 5'-start site. Clone 1 (accession number T65368) was 1779 bp, and clone 2 (accession number AA351071) was 1543 bp. Where the two
clones overlapped, their sequences were identical except that clone 2 had an in-frame deletion of 258 bp. To obtain the 5' start site, we
performed nested PCR on brain, lung, and liver cDNA libraries. The
longest amplified cDNA fragment contained 358 bp of additional 5'
sequence. We then searched the nonredundant data base of NCBI with
full-length SNX15 and identified an independent clone (AF001435) with
identical sequence at the 5' end. This clone (AF001435) had been
identified previously as an expressed gene on chromosome 11q13 during
the course of positional cloning of the gene for multiple endocrine
neoplasia type 1 (29). The coding sequence for the long form of SNX15
is 2061 bp, and the mRNA appears to contain at least 250 bp
upstream from the putative start site and 780 bp of 3'-untranslated
region including a poly(A) tail. The protein is predicted to contain
342 amino acid residues and to have a pI of 4.95 (Fig.
1A). SNX15 has 12 amino acid
residues at its N terminus followed by a 124-amino acid PX domain. The remaining 206 amino acids comprise the C terminus, which contains a
novel 73-amino acid region with homology to several protein families.
The first family encodes S. cerevisiae End13/Vps4 (30) and
its murine ortholog Skd1 (31) that have ~30% identity to this
region. The second family encodes Emericella nidulans PalB (32) and its yeast and human orthologs (Rim1p and PalBH, respectively) that have ~25% amino acid identity to this region (33, 34). We have
designated this region of homology as the ESP domain (for End13, SNX15,
PalB). The splice variant of SNX15, SNX15A, is predicted to contain 256 amino acid residues and have a pI of 6.21. It is identical to SNX15
except for the lack of 86 amino acid residues within the C terminus of
the molecule. Interestingly, SNX15A lacks a large portion of the ESP
domain (amino acids 222-308; Fig. 1B).
Both isoforms of SNX15 lack predicted transmembrane domains or leader
sequences and are expected to be soluble proteins. However, when we
examined the subcellular distribution of recombinant Myc-SNX15 and
Myc-SNX15A in COS7 cells, we found both isoforms in particulate, as
well as cytosolic fractions (Fig.
2A). Likewise, fractions prepared from nontransfected COS7 cells and probed with a polyclonal antibody to SNX15, detected SNX15 (~51 kDa) in both particulate and
cytosolic fractions (Fig. 2B), whereas the short isoform, SNX15A (~37 kDa), was not detected in either fraction.
Northern blots of numerous human tissues probed with a SNX15 cDNA
probe detected a broad band of ~2 kilobases. The highest levels of SNX15 mRNA expression were detected in skeletal muscle, heart, brain, kidney, spleen, thymus, and small intestine (Fig. 3). Because the conditions used for
northern analyses did not allow us to discriminate messages that
differed by only a few hundred nucleotides, we screened eight cDNA
tissue libraries by PCR using sequence-specific primers to determine
the expression pattern of SNX15 and SNX15A. Amplicons representing the
long isoform of SNX15 were observed in lung, liver, skeletal muscle,
prostate, pancreas, and adult and fetal brain. The short isoform,
SNX15A, was present in adult brain, liver, and placenta (data not
shown).
Association of SNX15 with Other Sorting Nexins--
Previously, we
showed that SNX1, SNX2, and SNX4 form homo- and hetero-oligomeric
complexes (4). To determine whether SNX15 could associate with itself
or other SNX molecules, COS7 cells were transiently transfected with
HA-tagged SNX15 alone or in combination with Myc-tagged SNX1, SNX2,
SNX3, SNX4, or SNX15. Total cell lysates were immunoblotted with
anti-Myc antibody to show the expression level of the various
recombinant Myc-tagged SNXs (Fig.
4C). Cell extracts were then
immunoprecipitated with anti-HA antibody and immunoblotted with an
anti-HA antibody. Similar amounts of HA-SNX15 were expressed and
immunoprecipitated in each sample (Fig. 4B). Reprobing the
same precipitates with an anti-Myc antibody showed that HA-SNX15
associates with itself (Fig. 4A, lane 5), and
with Myc-SNX1 (lane 1), Myc-SNX2 (lane 2), and
small amounts of Myc-SNX4 (lane 4). In contrast, HA-SNX15 did not
associate detectably with Myc-SNX3 (Fig. 4A, lane
3), although Myc-SNX3 was readily expressed (Fig. 4C,
lane 3). Because only small amounts of Myc-SNX4 were
coimmunoprecipitated with HA-SNX15, we designed confirmatory
experiments using a similar approach but switching the epitope tags
(Fig. 4, A-C, lane 7). HA-SNX4 was coexpressed with Myc-SNX15, cell lysates were immunoprecipitated with anti-Myc antibody, and the immune complexes were immunoblotted with an anti-HA
antibody. A strong band corresponding to HA-SNX4 was
coimmunoprecipitated with Myc-SNX15 (Fig. 4A, lane
7).
To determine whether the PX or ESP domains of SNX15 are required for
assembly into oligomers, we coexpressed several SNX15 mutants in
combination with SNX15. SNX15A is a naturally occurring splice variant
lacking the majority of the ESP domain (amino acids 222-308). Associations of SNX15 with Receptor Tyrosine Kinases--
SNX1,
SNX2, and SNX4 bind to receptor tyrosine kinases (4, 8). To investigate
whether SNX15 can also bind to growth factor receptors, Myc-SNX15 was
coexpressed in COS7 cells together with expression vectors encoding
receptors for insulin, EGF, or PDGF. Total cell lysates were
immunoblotted to show expression of the recombinant receptor proteins
(Fig. 6, bottom panel) and Myc-SNX15 (Fig. 6, middle panel, even lanes).
Although SNX15 was coimmunoprecipitated by antibody to the PDGF
receptor (Fig. 6, top panel, lane 2), we did not
detect association with receptors for EGF and insulin (Fig. 6,
top panel, lanes 4 and 6). We next sought to determine whether the tyrosine kinase activity of the PDGF
receptor was required for its association with SNX15. COS7 cells were
transiently cotransfected with Myc-SNX15 together with various forms of
the PDGF receptor: wild type, kinase inactive (K634R), or the F5 mutant
receptor in which five tyrosine phosphorylation sites were mutated to
phenylalanine (Y740F, Y751F, Y771F, Y1009F, and Y1021F) (15, 16).
Immunoblotting of cell extracts showed that Myc-SNX15 and the various
PDGF receptor proteins were all well expressed (Fig.
7, middle and bottom
panels). Cell extracts were also immunoprecipitated with antibody
directed against the PDGF receptor, and the immune complexes were
analyzed by immunoblotting with anti-Myc antibody (Fig. 7, top
panel). Small amounts of Myc-SNX15 associated with endogenous PDGF
receptors, despite their low levels of expression in COS7 cells (Fig.
7, top panel, lane 1). When PDGF receptors were
overexpressed, increased levels Myc-SNX15 were detected in
immunoprecipitates of the wild type PDGF receptor (Fig. 7, lane
2). In addition, Myc-SNX15 was coimmunoprecipitated with both the
kinase inactive and the F5 mutant PDGF receptor (Fig. 7, lanes
3 and 4). Addition of PDGF to the incubation medium did
not affect the association of SNX15 with the PDGF receptor (data not
shown). Taken together, these data suggest that SNX15 association with
the PDGF receptor is unchanged by activation of the PDGF receptor.
The PX Domain of SNX15 Is Required for Association with the
PDGFR--
To determine the region of SNX15 that is required for its
association with the PDGFR, we coexpressed our Myc-tagged SNX15 mutants
with the PDGF receptor. Cell lysates were immunoblotted, confirming
expression of Myc-tagged molecules (Fig.
8B) and the PDGF receptor
(Fig. 8C). Cell lysates were also immunoprecipitated with
antibody directed against the PDGF receptor followed by immunoblotting with anti-Myc (Fig. 8A). Deletion of the PX domain of SNX15
abolished association with the PDGF receptor (Fig. 8A,
lane 6). However, removal of some or the entire ESP domain
(lanes 3 and 4) or the entire C-terminal region
of SNX15 (lane 5) had no effect on the association of SNX15
with the PDGF receptor.
Overexpression of SNX15 Slows the Internalization and Degradation
of 125I-PDGF--
To investigate whether association of
SNX15 with the PDGF receptor affects the trafficking of the receptor;
we transiently transfected COS7 cells with recombinant PDGF receptors
in the absence or presence of coexpressed Myc-SNX15. After allowing the cells to bind 125I-PDGF for 4 h at 4 °C, the cells
were washed to remove unbound ligand and warmed to 37 °C for the
indicated times. At the end of the 37 °C incubation, the cells were
placed on ice, and the medium was collected. The medium was treated
with trichloroacetic acid to determine the amount of
125I-PDGF degraded (trichloroacetic acid-soluble
radioactivity) and the amount of intact 125I-PDGF
dissociated and/or recycled (trichloroacetic acid-precipitable radioactivity). The cells were then acid-washed to collect the 125I-PDGF bound to the cell surface, and the remaining
cell-associated counts were collected. In cells expressing only PDGF
receptors, 125I-PDGF was lost from the cell surface during
the warm-up period (Fig. 9A,
open symbols) and accumulated inside the cells during the
first 45 min at 37 °C (Fig. 9B, open symbols).
Starting after ~45 min at 37 °C, degraded ligand was detected in
the medium (Fig. 9C, open symbols) and continued
to increase for the remainder of the time course. During the last hour
at 37 °C, no change was seen in the amount of intracellular
125I-PDGF, indicating that a new steady state had been
reached (i.e. for each molecule internalized, a molecule was
degraded). In contrast, in cells coexpressing PDGF receptors and
Myc-SNX15, there was a marked decrease in the internalization of
125I-PDGF from the cell surface (Fig. 9, A and
B, solid symbols) and a 3-fold decrease in the
amount of 125I-PDGF degraded (Fig. 9C,
solid symbols).
Overexpression of SNX15 Impairs the Post-translational Processing
of the Insulin Receptor Precursor--
In the course of studying
whether SNX15 associated with receptor tyrosine kinases other than the
PDGF receptor, we consistently found that overexpression of SNX15 led
to a decrease in the amount of insulin receptor
To investigate whether SNX15 overexpression affected the localization
of furin, we transiently transfected COS7 cells with a tac-furin
chimera. The chimera consisted of the extracellular domain of the Structure of SNX15 The PX domain is the only structural feature shared by all sorting
nexins. This domain of ~100 amino acid residues was first identified
in the p40phox and p47phox subunits of NADPH
oxidase. To gain insight into the evolution of sorting nexins, we
constructed a dendrogram based upon amino acid sequence similarities
among PX domains contained in mammalian and yeast proteins (Fig.
14). Among the PX domains of known
sorting nexins, the SNX15 PX domain is most closely related to those of SNX1 and SNX2. However, whereas the homologies between SNX1 and SNX2
extend along the entire length of the molecules, the homology with
SNX15 is limited to the PX domain. Interestingly, another human
protein, BAA06542, also has a PX domain closely related to the PX
domain of SNX15. As is the case with SNX1 and SNX2, the homology
between SNX15 and BAA06542 is restricted to the PX domains. However,
based upon the sequence of its PX domain, BAA06542 belongs to the
family of sorting nexins and might be designated SNX16.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt10 universal
cDNA library (CLONTECH) as template. Standard molecular biology techniques were used to construct full-length cDNAs. The existence of each full-length SNX15 isoform was
confirmed by sequencing an additional full-length EST (accession number AF001435) and by PCR using tissue-specific cDNA libraries
(CLONTECH) and isoform specific primers. All
sequencing was performed using the ABI prism dye terminator cycle
sequencing kit and an ABI automated sequencer, model 373A (PerkinElmer
Life Sciences).
PX, lacks the N terminus
and PX domains of SNX15 (amino acids 1-135).
C lacks the entire C
terminus of SNX15 (amino acids 136-342), whereas the
ESP domain
lacks the entire ESP domain (amino acids 222-308) plus the remaining
34 C-terminal amino acids of SNX15. Epitope-tagged PCR products were
ligated into pcDNA3.1 (+) expression vector (Invitrogen, Carlsbad,
CA). The sequences of primers used to construct SNX15, SNX15A, and the
SNX15 mutant cDNAs are available upon request. pCIS2 expression
vector encoding full-length human receptor cDNAs for insulin (12),
EGF (13), and PDGF (14) have been previously described (12, 15). Mutant
human PDGF
-receptors were kindly provided by Dr. A. Kazlauskas. The
F5 mutant PDGF
-receptor cDNA contains substitutions of
phenylalanine for tyrosine at positions 740, 751, 771, 1009, and 1021 and is expressed in pCIS2 (15, 16). The kinase inactive PDGF
-receptor contains a K634R substitution (17) and was ligated into a
pCI-neo expression vector (Promega, Madison, WI). Human hepatocyte
growth factor (HGF) receptor cDNA was generously provided by Dr.
George Vande Woude (18). A SmaI/XhoI restriction
fragment containing the full-length receptor was ligated into pCIneo
expression vector. pCDM8.1 expression vector (Invitrogen) encoding the
interleukin-2 receptor
-subunit (tac) fused in frame with the
transmembrane and cytoplasmic domain of furin (tac-furin) was kindly
provided by Dr. Juan Bonifacino (19). The pEYFP-Golgi vector encoding the enhanced yellow fluorescent protein fused to the N-terminal 81 amino acids of the precursor to human
-1,4 galactosyltransferase was
obtained from CLONTECH.
-subunit of the insulin
receptor (C19), the EGF receptor (# 1005), the PDGF receptor
(
-isoform; # 958), the human HGF receptor (# 161), and c-Myc (A-14
and 9E-10) were obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Anti-HA antibody (HA.11) was from Covance (Richmond, CA).
ECL Western blot detection reagents, peroxidase-labeled donkey
anti-rabbit IgG, and peroxidase-labeled sheep anti-mouse IgG
were obtained from Amersham Pharmacia Biotech. Anti-SNX15 antibodies
were made by immunizing rabbits with a fusion protein consisting of
glutathione S-transferase and the coding region of SNX15.
The glutathione S-transferase fusion protein was expressed
using the pGEX-5x vector from Amersham Pharmacia Biotech. The
glutathione S-transferase fusion protein was purified from
Escherichia coli using glutathione-Sepharose (Amersham
Pharmacia Biotech). Rabbits were immunized at Covance Laboratories Inc.
(Denver, PA). Anti-tac antibody, clone 7G7/B6 (Upstate Biotechnology,
Lake Placid, NY) was used to detect the tac-furin chimera. The
secondary antibodies used for immunofluorescence were donkey IgG
conjugated to fluorescein isothiocyanate or conjugated to Rhodamine Red
(Jackson Immunoresearch Labs, West Grove, PA) or goat IgG conjugated to
Alexa 350 (Molecular Probes Inc., Eugene, OR).
-receptor with or without Myc-SNX15 expression vector. 24 h
later, the cells were washed twice and then incubated in binding buffer
(DMEM, 25 mM HEPES, pH 7.4, 0.5% w/v bovine serum albumin)
containing 125I-PDGF-2 (86.9 µCi/ml; PerkinElmer Life
Sciences) for 4 h at 4 °C. The media were removed; the cells
were washed again and then incubated in fresh binding buffer at
37 °C. After various times at 37 °C, the media were removed,
incubated with 12% (w/v) trichloroacetic acid overnight at 4 °C,
and then spun at (16,000 × g for 15 min) to separate
the trichloroacetic acid-insoluble counts (intact 125I-PDGF) from the trichloroacetic acid soluble counts
(degraded 125I-PDGF) present in the medium.
125I-PDGF present at the cell surface at each time was
determined by treating the cells twice with DMEM plus 25 mM
HEPES (pH 3.0) for 5 min at 4 °C to remove the cell surface counts.
Preliminary experiments showed that this acid wash procedure removes
>95% of the surface bound counts. Lastly, the cells were incubated for 30 min at 4 °C in PBS containing 1% (v/v) Triton X-100 and 1 mg/ml bovine serum albumin to collect the remaining cell associated counts (22), which were then treated with trichloroacetic acid (see
above). Each fraction was collected and counted (Autogamma Cobra II,
Packard Inc., Downers Grove, IL).
6, keeping known PX domain containing proteins and high
scoring unknowns in each round. The PX-containing regions of most
available human and Saccharomyces cerevisiae proteins were
aligned initially by clustal W (11), followed by manual editing,
removal of fragmentary and poorly alignable sequences, and trimming of
the poorly conserved ends of the alignment. The resulting curated
alignment of the ~100-amino acid residue core of the PX domain
sequences contained about 80 potentially informative positions for
phylogenetic analysis. This manually curated multiple alignment of PX
domain core region sequences was used for phylogenetic analysis, using
a variety of methods, including maximum parsimony (24), neighbor
joining with both PAM-based (25) and Poisson-corrected distance
measures in the PHYLO_WIN program (26) and both the Fitch-Margoliash (27) and neighbor joining in the Phyllip program (28). Clustering based
on sequence similarity rather than phylogenetics was performed by the
unweighted pair group method using arithmetic averages algorithm.2
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SNX15 is a novel PX domain-containing
protein. A, deduced amino acid sequence of human SNX15.
The coding sequence is shown in capital letters, and the 5'
and 3'-untranslated region are shown in lowercase letters.
The N-terminal PX domain is underlined, and the C-terminal
ESP homology domain is boxed. B, the long form of
human SNX15 (accession number AF175267) contains a PX domain (amino
acids 12-135) and an ESP homology domain (amino acids 265-337). The
short isoform, SNX15A (accession number AF175268) is identical to the
long isoform except that it lacks 86 amino acid residues (amino acids
222-308), including part of the ESP domain.
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Fig. 2.
Subcellular distribution of SNX15 and
SNX15A in COS7 cells. A, COS7 cells were transiently
transfected with Myc-tagged SNX15 (~51 kDa) or Myc-tagged SNX15A
(~37 kDa) followed 24-30 h later by preparation of total cell
lysates (T), particulate (P), and cytosolic
fractions (C), as described under "Experimental
Procedures." The distribution of Myc-SNX15 and Myc-SNX15A were
determined in each fraction by immunoblotting with an anti-Myc
antibody. B, subcellular distribution of endogenous SNX15 in
COS7 cells. Untransfected COS7 cells were plated and grown for 24 h followed by preparation of total cell lysates (T),
particulate (P), and cytosolic fractions (C). The
distribution of endogenous SNX15 was determined in each fraction by
immunoblotting with a polyclonal antibody generated against SNX15. The
long isoform (~51 kDa) was readily detected in COS7 cells, whereas
the short isoform with an expected molecular mass of ~37 kDa was not
detected.
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Fig. 3.
Tissue distribution of human SNX15
mRNA. Multiple human tissue Northern blots
(CLONTECH) containing poly(A)+ mRNA
and normalized for equal actin loading were hybridized with a
32P-labeled human SNX15 cDNA probe. The filters were
washed and exposed to film as described under "Experimental
Procedures."
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Fig. 4.
Association of human SNX15 with SNX1, SNX2,
SNX4, and SNX15. COS7 cells were transiently cotransfected with
expression vectors encoding HA-tagged SNX15 and Myc-tagged SNX1, SNX2,
SNX3, SNX4, or SNX15 as indicated. A, lanes 1-6,
total cell lysates were immunoprecipitated with anti-HA antibody,
separated by SDS-PAGE, and analyzed by Western blotting with an
anti-Myc antibody to detect coimmunoprecipitated SNX molecules.
B, lanes 1-6, The same blot was reprobed with
anti-HA antibody to determine the amount of HA-SNX15 (~51 kDa)
expressed and immunoprecipitated in each sample. C,
lanes 1-6, total cell lysates were analyzed by
immunoblotting with an anti-Myc antibody to determine the expression
levels of each Myc-tagged SNX. Because the SNX proteins vary in
molecular mass, the blots were cut and the Myc-positive bands are
shown. A similar experiment was performed using cells transiently
transfected with Myc-SNX15 and HA-SNX4. A, lane
7, total cell extract immunoprecipitated with anti-Myc antibody
and immunoblotted with anti-HA antibody. B, lane
7, same extract immunoblotted with an anti-HA antibody.
C, lane 7, result of reprobing of the blot in
A with an anti-Myc antibody. These experiments were repeated
three times with similar results.
PX
lacks the N terminus and PX domain of SNX15 (amino acids 1-135);
ESP lacks the entire ESP domain plus 34 remaining C-terminal amino
acids (), whereas
C lacks the entire C-terminal region of
the molecule (amino acid 136-342). SNX15A,
ESP, and
C SNX15 were
well expressed (Fig. 5A,
middle panel) and associated with SNX15 (Fig. 5A,
top panel, lanes 2-4). In contrast,
PX did not associate with SNX15 (top panel, lane 5) or
with SNX1, SNX2, or SNX4 (data not shown). Furthermore,
PX was found
almost exclusively in the cytosolic fraction (Fig. 5B),
whereas
ESP and
C SNX15 were distributed to both particulate and
cytosolic fractions like SNX15 and SNX15A (see Fig. 2).
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Fig. 5.
The PX domain of SNX15 is required for
self-association. A, COS7 cells were transiently
cotransfected with cDNAs encoding HA-SNX15 and various forms of
Myc-SNX15: empty vector (lane 1); Myc-SNX15A (lane
2); ESP, Myc-tagged SNX15 lacking amino acids 222-342
(lane 3);
C, Myc-tagged SNX15 lacking amino acids
136-342 (lane 4); and
PX, Myc-tagged SNX15 lacking amino
acids 1-135 (lane 5). Total cell lysates were
immunoprecipitated (IP) with anti-Myc antibody followed by
immunoblotting with anti-HA antibody to detect coimmunoprecipitated
HA-SNX15 (upper panel). The levels of expression of
Myc-SNX15A and the various Myc-SNX15 mutants are show in the
middle panel, and HA-SNX15 expression is shown in the
bottom panel. SNX15A is seen as a doublet. It is presently
unclear whether this is due to post-translational processing or
proteolysis of the molecule. This experiment was repeated three times
with similar results. B, COS7 cells were transfected with
the indicated SNX15 mutant cDNAs and 24-30 h later total cell
lysates (T), particulate (P), and cytosolic
fractions (C) were prepared as described under
"Experimental Procedures." The distribution of the various
Myc-tagged mutants of SNX15 was determined in each fraction by
immunoblotting with an anti-Myc antibody.
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Fig. 6.
SNX15 association with receptor tyrosine
kinases. COS7 cells were transiently transfected with mammalian
expression vectors for the receptors for PDGF (PDGFR,
lanes 1 and 2), insulin (IR,
lanes 3 and 4), or EGF (EGFR,
lanes 5 and 6) in the absence or presence of
recombinant Myc-SNX15. Total cell lysates were analyzed by
immunoblotting to detect expression of transfected cDNAs
(bottom and middle panels). In addition, total
cell lysates were immunoprecipitated (IP) with the
appropriate anti-receptor antibodies, separated by SDS-PAGE, and
analyzed by Western blotting using an anti-Myc antibody to detect
coimmunoprecipitated Myc-SNX15 (top panel). The insulin
receptor precursors (~210 and ~190 kDa) are indicated by
two and one asterisks, respectively. The mature
receptor -subunit is found at ~95 kDa. This experiment was
repeated three times with similar results.
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Fig. 7.
SNX15 association with the PDGF receptor does
not require an active tyrosine kinase. COS7 cells were transiently
transfected with cDNAs encoding Myc-SNX15 (lane 1) or
Myc-SNX15 and various forms of the PDGF receptor (PDGFR,
lanes 2-4): wild type (WT), kinase dead
(KD), or a mutant form of the PDGF receptor
(F5) with phenylalanine substituted for five of the
tyrosine phosphorylation sites. Total cell lysates were
immunoprecipitated (IP) with an anti-PDGF receptor antibody,
followed by immunoblotting to detect coimmunoprecipitated Myc-SNX15
(top panel). Expression of Myc-SNX15 is demonstrated in the
middle panel, and the expression of the various PDGF
receptors in the bottom panel. This experiment was repeated
three times with similar results.
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Fig. 8.
The PX domain of SNX15 is required for
association with the PDGF receptor. COS7 cells were transiently
cotransfected with cDNAs encoding PDGF receptor and various forms
of Myc-SNX15. Myc-SNX15 alone (lane 1); the PDGF receptor
plus Myc-SNX15 (lane 2), Myc-SNX15A (lane 3); ESP, SNX15
lacking amino acids 222-342 (lane 4);
C, SNX15 lacking
amino acids 136-342 (lane 5); and
PX, SNX15 lacking
amino acids 1-135 (lane 6). Total cell lysates were
immunoprecipitated (IP) with anti-PDGF receptor antibody
followed by immunoblotting using anti-Myc antibody (top
panel). The levels of expression of the various forms of SNX15 are
shown in the middle panel, and the level of PDGF receptor
expression is shown in the bottom panel. These experiments
were repeated two times with similar results.
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Fig. 9.
Internalization and degradation of
125I-PDGF. COS7 cells were transfected with a cDNA
encoding the PDGF receptor alone (open squares) or with
Myc-SNX15 (filled squares). After 24 h,
125I-PDGF was bound at 4 °C for 4 h. The cells were
washed at 4 °C to remove unbound ligand and then warmed to 37 °C.
At the indicated times, the medium was removed, treated with
trichloroacetic acid, and centrifuged to separate the trichloroacetic
acid precipitable 125I-PDGF (intact ligand in the medium)
from the trichloroacetic acid-soluble 125I (degraded ligand
in the medium). The cells were then washed and incubated with a low pH
buffer to remove surface bound 125I-PDGF. The remaining
cell associated radioactivity (internalized ligand) was quantified by
solubilizing the cells in Triton X-100 containing buffer (22), which
were then treated with trichloroacetic acid (see above). Each fraction
was collected and counted. A, the y axis shows
the percentage of the initial counts bound at 4 °C that remained at
the cell-surface following various times of warm up. B, the
y axis shows the percentage of the initial counts that were
found intact inside the cell. C, the y axis shows
the percentage of the initial counts that were degraded. Results are
presented as the means ± S.E. for three time courses where each
time point was performed in triplicate.
-subunit present in
COS7 cells (Fig. 6A, bottom panel). The insulin
receptor is synthesized as a 190-kDa pro-receptor containing
N-linked high mannose oligosaccharides (35). The
N-linked oligosaccharides undergo processing to yield complex oligosaccharides and an increase in molecular mass to 210 kDa (36). Cleavage of the 210-kDa precursor protein by the endoprotease furin results in mature
- (135 kDa) and
-subunits (95 kDa) (Fig. 10A)
(36-38). To investigate whether the decrease in mature insulin
receptor
-subunit seen in cells overexpressing SNX15 might be due to
impairment in the post-translational processing of the receptor
precursor, we performed pulse-chase studies in COS7 cells. After
labeling for 20 min with [35S]cysteine plus
[35S]methionine, transfected cells were chased for 0-20
h in complete medium containing excess unlabeled cysteine and
methionine. Cell lysates were then immunoprecipitated with anti-insulin
receptor antibody and analyzed by SDS-PAGE followed by autoradiography (Fig. 10B). In cells expressing only recombinant insulin
receptors (Fig. 10B, open boxes), the 190-kDa
pro-receptor accumulates, attaining a peak level ~4 h into the chase.
Moreover, the 210-kDa form of the receptor is present only at low
levels, suggesting that the pro-receptor undergoes efficient
proteolytic processing by furin to the mature mature
- and
-subunits. In contrast, when SNX15 was coexpressed, this led to an
increase in the levels of both the 190-kDa precursor and the 210-kDa
species (Fig. 10B, solid boxes, left
and middle panels, respectively) with a corresponding decrease in the level of mature
-subunit (Fig. 10B,
right panel, solid boxes). Similar delays in
pro-receptor processing were seen by Western blotting of total cell
extracts from cells overexpressing both SNX15 and the insulin receptor
(Fig. 11A, lanes 1 and
2). Interestingly, overexpression of SNX15 also delayed the
post-translational processing of the HGF receptor, another substrate of
the endoprotease furin (Fig. 11B). When COS7 cells were
transfected with HGF receptors in the presence of recombinant
Myc-SNX15, there was a 2-fold increase in the immunodetectable 170-kDa
HGF pro-receptor and a corresponding decrease in the 145-kDa mature
-subunit as compared with cells expressing the HGF receptor alone
(Fig. 11B, lanes 1 and 2). In contrast, when COS7 cells were transiently transfected with a cDNA
encoding the PDGF receptor (a receptor that does not undergo furin-dependent processing) in the presence or absence of
Myc-SNX15, there was no change in the immunodetectable precursor or
mature form of the PDGF receptor (Fig. 6, bottom panel).
These findings suggest that SNX15 overexpression interferes with the
activity and/or localization of furin.
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Fig. 10.
Overexpression of SNX15 slows processing of
the insulin receptor. A, the insulin receptor is
synthesized as a 190-kDa precursor. Its high mannose
N-linked oligosaccharides undergo processing to yield
complex oligosaccharides leading to an increase in the molecular mass
of the precursor to 210 kDa. Subsequently, the 210-kDa precursor is
cleaved by furin to yield mature - and
-subunits. B,
pulse-chase labeling studies. COS7 cells were transfected transiently
with an expression vector for the insulin receptor in the presence
(filled squares) or absence (open squares) of
expression vector for Myc-SNX15. Cells were pulse labeled for 20 min
with [35S]cysteine + [35S]methionine and
then chased for 0-20 h in complete medium containing excess cysteine
and methionine. Cell lysates were immunoprecipitated with anti-insulin
receptor antibody. Immune complexes were analyzed by SDS-PAGE and
transferred to nitrocellulose. A PhosphorImager was used to quantify
the radioactivity in the bands corresponding to the 190-kDa
pro-receptor, the 210-kDa precursor, and the 95-kDa
-subunit. These
data, expressed in arbitrary units, are plotted as a function of
time.
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Fig. 11.
Expression of SNX15 impairs
post-translational processing of the insulin receptor and the HGF
receptor. A, COS7 cells were transiently transfected
with a cDNA encoding the insulin receptor in the absence or
presence of recombinant Myc-SNX15. Cell lysates were analyzed by
SDS-PAGE, followed by immunoblotting with an anti-insulin receptor
antibody to detect the insulin receptor precursors (pre-,
210 and 190 kDa), and the mature -subunit (95 kDa). An anti-Myc
antibody was used to detect recombinant Myc-SNX15. B, COS7
cells were transiently transfected with a cDNA encoding the HGF
receptor in the absence or presence of recombinant Myc-SNX15. Cell
lysates were analyzed by SDS-PAGE, followed by immunoblotting with an
anti-HGF receptor antibody to detect the receptor precursor
(pre-, 170 kDa), and the mature
-subunit (145 kDa). An
anti-Myc antibody was used to detect recombinant Myc-SNX15.
subunit of the interleukin-2 receptor fused to the transmembrane and
cytoplasmic domains of furin (19). It has previously been shown that
both native furin and the tac-furin chimera are localized predominantly
in the TGN. However, both molecules continually cycle from the TGN to
the plasma membrane and then back to the TGN via endosomes (19, 39).
When tac-furin was coexpressed with a marker for medial and
trans-Golgi, i.e. the enhanced yellow fluorescent
protein fused to 81 N-terminal amino acids of human
-1,4-galactosyl
transferase; EYFP-GT, both molecules were detected in perinuclear Golgi
structures (Fig. 12, B and
C). At higher levels of expression, the tac-furin chimera was also found in small puncta, presumably endosomes, through which
furin normally cycles. Endogenous COS7 SNX15 was found in the cytoplasm
and also in many small punctate structures (Fig. 12A). In
the merged images, the TGN of the transfected cells is stained aqua,
indicating colocalization of tac-furin and EYFP-GT. When COS7 cells
also overexpressed recombinant Myc-SNX15, the EYFP-GT staining of the
TGN was unchanged (Fig.
13C). However, the
localization of tac-furin was altered (Fig. 13B). In
addition to being detected in the TGN (indicated by
arrowheads), tac-furin was found in rings, fused rings, and
larger aggregates (Fig. 13B). Recombinant Myc-SNX15 was also
seen in these aberrant structures rather than in small puncta (Fig.
13A). The merged images show that some tac-furin remains
localized to the Golgi with EYFP-GT (Fig. 13D,
aqua). However, there is now colocalization of tac-furin and
SNX15 in the aberrant structures (Fig. 13D,
magenta). Taken together, these data suggest that
overexpression of SNX15 altered the steady state localization of furin,
resulting in delayed processing of several furin substrates.
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Fig. 12.
Localization of tac-furin and endogenous
SNX15. COS7 cells were transfected with tac-furin and a Golgi
marker, EYFP-GT. After 20 h the cells were fixed, permeabilized,
and stained. A, cells were stained for endogenous SNX15 with
a rabbit anti-SNX15 primary antibody and visualized with a Rhodamine
Red-conjugated donkey anti-rabbit IgG secondary antibody
(red). B, cells were also stained for tac-furin
with mouse anti-tac primary antibody and visualized with an Alexa 350 conjugated goat anti-mouse IgG secondary antibody (blue).
C, cells expressing EYFP-GT, a medial and
trans-Golgi marker, are shown (green). Endogenous
SNX15 is seen in the cytoplasm and in numerous small puncta
(A, red). Recombinant tac-furin is located
predominantly in perinuclear structures, and at higher levels of
expression, the chimera is also found in small puncta, presumably
endosomes, through which it normally cycles (B,
blue). As expected, EYFP-GT is seen in brightly stained
perinuclear Golgi structures (C, green).
D, in the merged image, the TGN is stained aqua,
indicating colocalization of tac-furin and EYFP-GT. In addition,
the cell expressing high levels of tac-furin shows some colocalization
of tac-furin with SNX15 in small puncta (violet).
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Fig. 13.
SNX15 overexpression results in
mislocalization of tac-furin. COS7 cells were transfected with
Myc-SNX15, tac-furin, and EYFP-GT. After 20 h the cells were
fixed, permeabilized, and stained. A, cells were stained for
Myc-SNX15 with a rabbit anti-Myc primary antibody and visualized with a
Rhodamine Red-conjugated donkey anti-rabbit IgG secondary antibody
(red). B, cells were also stained for tac-furin
with mouse anti-tac primary antibody and visualized with an Alexa 350 conjugated goat anti-mouse IgG secondary antibody (blue).
C, cells expressing EYFP-GT are shown (green).
Recombinant Myc-SNX15 is seen in rings, fused rings, and larger
membrane-limited structures rather than in small puncta (A,
red). Although some recombinant tac-furin remains in
perinuclear structures, in cells overexpressing SNX15, the majority of
the chimera is now colocalized in SNX15-positive aberrant structures
(B, blue). In contrast, EYFP-GT staining is
unchanged by Myc-SNX15 overexpression (C, green).
D, the merged images show that some tac-furin remains
localized to the Golgi with EYFP-GT (indicated by arrowheads
and aqua color). However, the majority of the tac-furin
staining is now colocalized in rings, fused rings, and larger
SNX15-containing aggregates (violet).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
In this study, we have identified and
characterized a novel sorting nexin, SNX15. Like other sorting nexins, SNX15 contains a PX domain. We show here that the PX domain of SNX15 is
required for its self-association, association with other sorting
nexins, and its association with the PDGF receptor. Deletion of the PX
domain from SNX15 not only blocks its associations with other molecules
but also prevents association of SNX15 with intracellular membranes. We
have also identified a new homology domain in the C terminus of SNX15
(amino acids 265-337), designated the ESP domain. The ESP domain is
found in two known proteins and three as yet uncharacterized genes from
Drosophila and human. Fungal PalB and its yeast ortholog,
Rim1p, are involved in a conserved signal tranduction cascade mediating
adaptive responses to changes in pH (32, 33). Yeast End13p/Vps4p and
its mammalian ortholog Skd-1 (31) are members of the AAA ATPase family
of proteins important in movement of proteins from the prevacuolar
endosome (30, 40, 41). The function of the ESP domain is presently unknown. However, the N-terminal portion of the domain shares many
characteristics with the tetratricopeptide repeat homology (TPR)
consensus sequence. A region of the ESP domain is 32% identical and
53% similar to the first TPR of protein phosphatase 5, for which the
crystal structure is known. Each TPR forms a structural element with
two anti-parallel
- helices joined by a turn. Tandem TPR domains
stack into structures that may be involved in protein-protein interaction. The ESP domain may contain a similar pair of anti-parallel
- helices but in a different structural context (42).
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Fig. 14.
Phylogenetic tree of PX-containing
proteins. A phylogenetic tree of PX-containing proteins based on a
multiple alignment of the PX domains. Columns of the alignment
containing gaps in a least one sequence were ignored, leaving 80 columns. The PAM (25) model of mutational distance was used for the
neighbor-joining algorithm of tree construction by PHYLO_WIN (26). The
branch that contains p40phox, p47phox, and phosphoD2, among others, was
selected as an outgroup, which roots the tree. SNX15 can be seen to
descend from the same node as the last common ancestral node of SNX1,
Vps5p, Grd19p, and other sorting molecules. The GenBankTM
protein accession numbers for yeast proteins are as follows: Vam7p
(AAC4949), Vps5 (AAB62976), Vps17p (NP 014775), Grd19p (NP015002),
Mdm1p (NP013603), Mvp1p (NP013717), YDL113c (CAA98681), YDR425w (NP
010713), YJL036w (CAA89327), and YBR200w (CAA85163). The
GenBankTM protein accession numbers for human proteins are
as follows: SNX1 (AAC17182), SNX2 (AAC17181), SNX3 (NP003786), SNX4
(AAC83149), SNX5 (AAD27828), SNX6 (AAD27829), SNX7 (AAD27830), SNX8
(AAD27831), SNX9 (AAD27832), SNX10 (AAD27833), SNX11 (AAD27834), SNX12
(AAD48491), SNX14 (AAD27836), SNX15 (AF175267), p40phox
(BAA89792), p47phox (AAB64186), phosphatidylinositol
3-kinase (JC5500), I-1 receptor candidate protein (AAC33104), BAA06542,
and AAD32668. The SNX13 PX domain fragment is not included in the
tree.
Previously, we had concluded that the PX domains of SNX1, SNX2, SNX3, and SNX4 were more closely related to one another than to PX domains in other proteins. Indeed, this contributed to the rationale for defining sorting nexins as a subgroup of PX domain-containing proteins. We have now extended this conclusion to a larger group of 31 PX domain containing proteins in yeast and mammals. With the exception of SNX14, the dendrogram suggests that there is a close evolutionary relationship among the PX domains of all the sorting nexins in the data base. In this analysis, SNX14 does not segregate in the group of proteins containing other sorting nexins or their yeast orthologs. Rather, SNX14 lies outside the group of sorting nexins together with a number of other proteins including the p40phox and p47phox subunits of NADPH oxidase, class II phosphatidylinositol 3-kinase, and phospholipase D2. This raises questions about the appropriateness of the designation SNX14.
In yeast, several PX domain-containing proteins are involved in protein
trafficking. For example, Vps5p, the S. cerevisiae ortholog
of SNX1, is a subunit of a multimeric complex termed "the retromer
complex" that is involved in recycling of the carboxypeptidase Y
receptor from endosomes to the TGN (5, 9, 43). Grd19, the yeast
ortholog of SNX3, is required to maintain the steady state localization
of two late-Golgi enzymes (dipeptidyl amino peptidase A and Kex2) by
retrieving mislocalized molecules from prevacuolar endosomes (7). In
addition, Mvp1p, the yeast ortholog of SNX8, is thought to function in
the formation of transport vesicles that facilitate vacuolar protein
targeting (6). Here we show that overexpression of SNX15 affects at
least two membrane trafficking events. Excess SNX15 decreases the
internalization and degradation of the PDGF receptor and also delays
the post-translational processing of the pro-receptors for insulin and
HGF into their mature subunits. Because direct binding of SNX15 to the
insulin receptor (Fig. 6) or
tac-furin3 was not
demonstrated, it is unlikely that the above noted effects result from a
direct interaction of the molecules. Rather, our findings are most
likely due to the effects of SNX15 overexpression on endosome structure
and function. Our immunofluorescence studies also show that
overexpression of SNX15 leads to mislocalization of furin and
inefficient processing of furin substrates. This SNX15-induced defect
in processing of the insulin receptor precursor can be partially
corrected by overexpressing additional furin.3 These
findings, as well as studies in yeast, suggest a possible mechanism for
the observed defect in insulin receptor processing. Yeast Kex2p is a
furin-like protease. Retention of Kex2p in its normal location in the
Golgi is dependent upon Grd19p, the yeast ortholog of SNX3. It is
possible that overexpression of SNX15 disrupts a similar sorting
nexin-dependent pathway required to retain furin in the
TGN, thereby disrupting proteolytic processing of the insulin receptor.
Recent findings from our laboratory support this idea. In addition to
disrupting the normal trafficking of furin, we have shown that SNX15
overexpression also disrupts the endocytosis and normal trafficking of
transferrin and of TGN38, another protein that cycles between the TGN,
plasma membrane, and endosomes. SNX15 overexpression very dramatically
affects the morphology of the endocytic pathway leading to the
formation of membrane-limited structures containing markers for early
endosomes, late endosomes and lysosomes but not markers of the
secretory pathway (44). Taken together these findings clearly
demonstrate that SNX15 is involved in protein trafficking. At present,
we do not know whether SNX15 interacts with TIP47 (45) or PACS-1 (46)
that are also involved in retrograde trafficking in mammals. SNX15 and
the many other members of the sorting nexin family of proteins share in
common the ability to bind to receptor tyrosine kinases, to
self-associate, to associate with each other, and to associate with
intracellular membranes (4). Further studies are necessary to define
the exact functions that each of these molecules has in directing
protein trafficking in mammalian cells.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Bernard Desbuquois for critical reading of the manuscript. We also thank Dr. Andrius Kazlauskas for the generous gift of PDGF receptor cDNA, Dr. George Vande Woude for the HGF receptor cDNA, and Dr Juan Bonifacino for the tac-furin cDNA. In addition, we thank George Poy for DNA sequencing. Finally, we thank Lucy de la Luz Sierra, Nicola Perrotti, Ruth He, and Jill Sherman for contributions to this project.
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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.
¶ To whom correspondence should be addressed: Diabetes Branch, NIDDK, National Institutes of Health, Bldg. 10, Rm. 8N244, Bethesda, MD 20892. Tel.: 301-594-7689; Fax: 301-402-0573; E-mail: carol_ haft{at}nih.gov.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M004671200
2 E. Sonnhammer, unpublished data.
3 S. A. Phillips, S. I. Taylor, and C. Renfrew Haft, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
SNX, sorting nexin;
EGF, epidermal growth factor;
PDGF, platelet-derived growth factor;
TGN, trans-Golgi network;
PX, phox homology;
EST, expressed
sequence tag;
HA, influenza hemagglutinin;
HGF, hepatocyte growth
factor;
DMEM, Dulbecco's modified Eagle's medium;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
ESP domain, End13, SNX15, PalB homology domain;
TPR, tetratricopeptide
repeat;
EYFP-GT, enhanced yellow fluorescent protein -1,4
galactosyltransferase;
bp, base pairs;
PCR, polymerase chain
reaction.
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REFERENCES |
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