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INTRODUCTION |
Rab GTPases are a family of 20-29-kDa Ras-like GTPases localized
to unique intracellular organelles (1-7). They cycle through an active
membrane-bound GTP- and inactive cytosolic GDP-bound states. They
contain a characteristic Cys-containing motif at the extreme carboxyl
terminus that is post-translationally modified by one or more prenyl
residues (8-13). This modification is essential for membrane
localization and function. The GDP-bound Rab is removed from the
membrane by a cytosolic carrier protein known as GDI or GDP
dissociation inhibitor, which also inhibits the dissociation of GDP and
maintains Rab in the inactive state. Guanine nucleotide exchange occurs
at the membrane and is catalyzed by a guanine nucleotide exchange
factor of which a Rab3-specific (14) and the yeast
Sec4p-specific form have been identified (15, 16). The low intrinsic
GTPase activity requires catalysis by a GTPase activating protein of
which a Rab3A-specific (17) and yeast Ypt-specific (18, 19) forms have
been identified.
In ER1 to Golgi membrane
trafficking, donor vesicles are tethered to the acceptor membrane
compartment prior to SNARE-mediated fusion (20-23). Rab has been shown
to mediate the recruitment of a cytosolic tethering protein, Uso1p, to
the membranes. Another large protein complex called TRAPP may also be
involved in this transport step (24). Tethering factors have been
identified in other transport steps, suggesting conservation of this
basic underlying process (25-27). Once docked, however, vesicle fusion is no longer dependent on Rab, but requires the SNARE proteins. Thus,
stability of docked vesicles may represent a number of distinct molecular states: from molecular interactions that merely hold the two
membranes in close proximity to those needed to trigger bilayer fusion.
Studies on synaptic vesicles have shown that docked vesicles will
undock about once every 2 min, a rate that is faster than spontaneous
fusion (28). Thus, reversibility of vesicle tethering and docking
provides a time constraint on subsequent steps.
Little is known on the mechanism of Rab localization to a specific
membrane compartment. We have recently isolated a Rab-interacting protein called prenylated Rab
acceptor or PRA1 (29). PRA1 interacts with both Rab and
VAMP2 but not as a stable trimeric complex. In fact, binding of PRA1 to
VAMP2 can be displaced by Rab3A, suggesting that PRA1 participates in
the sequential assembly of protein by associating and dissociating from
the Rab and VAMP2. Moreover, PRA1 inhibits the removal of Rab from the
membrane by GDI (30). Thus, the recycling of Rab depends on the
opposing action of PRA1 and GDI, with PRA1 favoring membrane retention
and GDI favoring solubilization in the cytosol.
The existence of multiple isoforms seems to be a common theme in
proteins involved in the various vesicle transport steps. Multiple
isoforms of VAMP and syntaxin supports the concept that distinct
members mediate specific membrane trafficking steps (31-33). We
describe here the isolation of a second PRA isoform that shares many
similarities to PRA1 including overall physical properties, tissue
distribution, and broad binding specificity toward the Rab GTPases.
However, it differs from PRA1 in its subcellular localization. Whereas
PRA1 was localized predominantly in the Golgi complex, PRA2 was found
in the ER compartment. Moreover, we showed that the localization signal
resides in the COOH-terminal region of the proteins.
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EXPERIMENTAL PROCEDURES |
Isolation of PRA2--
A EST data base search using the
conserved domain of the rat PRA1 resulted in a number of similar
clones, most notably that with accession numbers AW519501 and AA051031.
The rat PRA2 was subcloned into a vector containing the hemagglutinin
(HA) tag by ligation between the StuI and XbaI
sites. PRA2 was PCR-amplified from a rat brain cDNA library using
the following oligonucleotides: 5'-TAAGGCCTATGGACGTGAACCTCG-3' and
5'-GCTCTAGATTACTCCCTCGCTTTGCTGA-3'. The resulting PCR fragment was sequenced.
Northern Blot Analysis--
A 32P-labeled
full-length PRA2 probe was prepared using random hexamer labeling (Life
Technologies). A multiple tissue blot (CLONTECH)
containing poly(A)+-selected mRNA of various rat
tissues was hybridized with 1 × 106 cpm/ml of
32P-labeled PRA2 in 10 ml of hybridization solution (5 × SSPE, 10 × Denhardt's solution, 100 µg/ml sheared salmon
sperm DNA, 50% formamide, and 0.4% SDS) at 42 °C overnight.
Plasmid Construction--
PRA2 was subcloned into the yeast
two-hybrid prey vector, pGAD424X using PRA2.E,
5'-CGAGAATTCTTACTCCCTGGCTTTGCTGAT-3' and PRA2.X2,
5'-GCCTCGAGTTACTCCCTSGCTTTGCTGA-3'. The VAMP1, VAMP2, and VAMP3
bait plasmids were described previously (29).
The HA-tagged PRA2 was subcloned into pQE10 (Qiagen) between the
BamHI and the HindIII sites after PCR
amplification using the following oligonucleotides:
5'-GCGGATCCTATGTACCCATACGATG-3' and 5'-GACAAGCTTACTCCCTGGCTTTGC-3'. The
6xHis-tagged PRA2 was purified the same way as the PRA1 using Ni-NTA
resin as described (29).
The Rab GTPases were also subcloned into the 6xHis-tagged pQE41
(Qiagen) between the BamHI and SphI sites. Rab1A
was PCR-amplified using the following oligonucleotides:
5'-TGTGGATCCATGTCCAGCATGAATCCCGAA-3' and
5'-CTAGGCATGCTTAGCAGCAGCCTCCACCTG-3'. The 6xHis-tagged Rab1A fragment
was ligated into pYES2 (Invitrogen) between the EcoRI and
SphI sites. The plasmid was then transformed into the yeast INVSc1 strain with Li acetate (34). To purify the 6xHis-tagged Rab1A,
the transformed yeast was grown to saturation in 500 ml of Ura dropout
medium. The cells were harvested at 3,000 × g for 5 min, transferred to 1 liter of YPG (containing 2% galactose and 1%
raffinose instead of glucose), and grown at 30 °C for 8 h. The
Rab1A was then purified from the yeast as described previously for
Rab3A (30), except that phosphate-buffered saline was used instead of
Tris-HCl and the proteins were eluted from the resin with 50 mM EDTA. The purified Rab3A and Rab1A were cross-linked to
CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech AB) as
described by the manufacturer.
Various domains of HA-tagged wild-type PRA1 and PRA2 were PCR amplified
using pIRES/HA-PRA1 and pIRES/HA-PRA2 as DNA templates. Two rounds of
PCR were used to generate the chimeras. The first round of PCR
generated the individual PRA1 or PRA2 fragments, which served as
templates for the second round to create the different chimeras. The
PCR products were digested with ClaI and EcoRI
and subcloned into the same restriction sites of the mammalian
expression vector pIRESpuro (CLONTECH). The
carboxyl regions of both PRA1 and PRA2 were PCR-amplified using the
Bluescript KSII(
)/HA-PRA1 and KSII(
)/HA-PRA2 as DNA templates. PCR
products were gel purified and subcloned into the GFP-containing
vector, pEGFP-C1 (CLONTECH) as XhoI and
BamHI fragments such that the GFP was fused to the amino
terminus of the PCR product. All plasmids were sequenced to confirm the
expected mutations.
Rab Binding Assay--
The binding of Rab1A and Rab3A to PRA1
and PRA2, in the presence of GDP
S or GTP
S was done using purified
yeast or bacterially expressed proteins in a pull-down assay. A typical
binding assay contained 20 µl of 50% bead slurry cross-linked with
Rab1A or Rab3A at 2 pmol/µl. This was incubated for 1 h at
4 °C with 10, 15, and 20 pmol of recombinant PRA1 or PRA2 in a total
volume of 250 µl of 25 mM Tris-HCl, pH 7.5, 125 mM KCl, 0.005% Triton X-100, 10% glycerol, 0.5 mM MgCl2, and 250 µM GDP
S or
GTP
S (Roche Molecular Biochemicals). Controls were also done using
Sepharose beads with no cross-linked Rab. The beads were then washed
three times with 1 ml of ice-cold wash buffer (25 mM
Tris-HCl, pH 7.5, 125 mM KCl, and 0.005% Triton X-100,
10% glycerol). Denaturing loading buffer was added to the beads and
proteins were subjected to Western immunoblot analysis. PRA1 and PRA2
were detected using anti-HA antibodies (Roche).
Transfection, Subcellular Fractionation, and
Immunocytochemistry--
All plasmid constructs were transfected into
Chinese hamster ovary (CHO) cells using LipofectAMINE (Life
Technologies). CHO cells were seeded at 1 × 105 on
12-mm diameter coverslips overnight and transfected with 0.5 µg of
DNA in 1.5 µl of LipofectAMINE. The cells were fixed after 36-48 h
in 4% paraformaldehyde (EM Sciences) in phosphate-buffered saline for
30-60 min and washed with 100 mM glycine in
phosphate-buffered saline. Cells were incubated with blocking buffer
(1% bovine serum albumin, 2% normal goat serum, and 0.4% saponin in
phosphate-buffered saline) for 30-60 min at room temperature. Mouse
monoclonal anti-HA antibodies (Roche Molecular Biochemicals) were
diluted in blocking buffer and incubated with the cells for 1 to 2 h at room temperature. Cells were then washed with 100 mM
glycine in phosphate-buffered saline and Alexa 488- or Alexa
568-labeled secondary antibodies (Molecular Probes) were used to detect
the bound primary antibodies. Rabbit anti-calnexin (Stressgen) and
anti-mannosidase-II (kindly provided by Dr. M. G. Farquhar) were
used together with the monoclonal anti-HA in certain cases. Coverlips
were mounted with Slow Fade anti-quench solution (Molecular Probes) and
confocal laser microscopy (Bio-Rad MRC-1024MP) was used to capture the
images. The cells were fixed and stained to visualize the chimeric
constructs; however, live cells were imaged for the GFP fusions.
For subcellular fractionation, 100-mm plates of CHO cells were
transfected with pIRESpuro/HA-PRA2 in LipofectAMINE. After 24-48 h,
the cells were harvested and homogenized in 1 ml of 10 mM
Tris-HCl, pH 7.5, 150 mM NaCl (TBS) supplemented with 2 mM phenylmethylsulfonyl fluoride. The crude homogenate was
spun at 5,000 × g for 10 min to remove the cell
debris, and the resulting supernatant was subjected to centrifugation
at 100,000 × g for 1 h to yield the high speed
cytosolic and intracellular membrane fractions. To further characterize
the membrane-associated PRA2, the high speed membrane fractions were
resuspended in 0.1 ml of TBS and extracted with 20 volumes of 0.1 M sodium carbonate, pH 11.5, at 4 °C for 30-60 min. The
membranes were then collected by centrifugation at 100,000 × g for 1 h (4 °C). For Triton X-114 extraction, the
resuspended membranes were solubilized in 1% Triton X-114 at 4 °C
for 30-60 min, and induced to phase partition at 37 °C for 10-15
min followed by centrifugation at 10,000 × g for 5 min
at room temperature. The resulting detergent phase was back extracted
three times with 10 volumes of ice-cold TBS at 4 °C for 10 min, and
phase partitioned as before. The resulting supernatants were pooled and
precipitated with 10% trichloroacetic acid.
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RESULTS |
Isolation and Structural Features of PRA2--
Proteins involved
in vesicle transport often exist as multiple isoforms with each
mediating specific membrane trafficking steps. A BLAST search of the
mouse and rat EST data bases with the conserved region of rat PRA1
revealed a number of overlapping clones. Merging the sequences from two
of the longest mouse clones (accession numbers AW519501 and AA051031)
revealed an open reading frame of 564 nucleotides. Oligonucleotides
spanning the initiation and termination codons were used to PCR amplify
rat and mouse brain cDNA libraries. Both gave a PCR product of the expected size, and were subsequently subcloned into a HA-tagged Bluescript vector for DNA sequence analysis. The open reading frame of
the rat PRA2 contained 188 amino acids with a predicted molecular mass
of 21.5 kDa. As with PRA1, PRA2 also contained two extensive
hydrophobic domains, of 36 and 35 residues spanning amino acids 47 to
82 and 101 to 135, respectively. Comparison of the two rat PRA
sequences revealed an overall amino acid identity of 26% and a
similarity of 35% (Fig. 1). The most
conserved domain was located at residues 35 to 47, immediately
amino-terminal to the first hydrophobic segment of the two proteins.
One notable difference between the two proteins was the length of the
amino- and COOH-terminal domains flanking the two hydrophobic segments. PRA2 has a shorter amino-terminal domain of 46 residues compared with
77 in PRA1, but a longer COOH-terminal domain of 53 residues compared
with 21 in PRA1. The PRA2 COOH-terminal domain has a cluster of basic
residues in contrast to PRA1 which has a number of acidic residues.
Moreover, this COOH-terminal domain of PRA2 has a weak coiled-coil
conformation, which might play a role in protein-protein interaction. A
search of the data base revealed that PRA2 is identical to the rat JWA
(accession number AAF60354) and highly similar to the human JM4
(accession number NP 009144) clones.

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Fig. 1.
PRA2 sequence and comparison to PRA1.
The sequences were compared using BestFit alignment program with
identical residues indicated by a bar and similar residues
with a colon. The predicted hydrophobic domains are
underlined.
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Northern blot analysis showed that PRA2 was encoded by a single
1.5-1.8-kilobase transcript and broadly expressed in most tissues
(Fig. 2). The message was most abundant
in the heart and brain. This expression pattern was similar to that of
PRA1 (29) with one exception: there was a lower level of expression of
PRA2 in the testis whereas PRA1 was highly expressed. This broad
expression pattern suggests that PRA2 might also be involved in
membrane trafficking events in all tissues. In both PRA1 and PRA2, the testis transcript appeared larger than that from other tissues. The
reason for this remained unknown.

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Fig. 2.
Northern blot analysis of PRA2.
Poly(A)+-selected mRNA was probed with the entire
coding sequence of PRA2 labeled with 32P. The position of
the RNA standards (in kilobase) is indicated on the
left.
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PRA2 Binds Multiple Rab Isoforms in a Guanine
Nucleotide-independent Manner--
Since PRA1 can bind both GDP- and
GTP-bound Rab, we examined the interaction of PRA2 with the Rab
GTPases. In the yeast two-hybrid system, PRA2 showed a positive
-galactosidase reaction when tested against the wild-type and GTPase
mutant Rab1A and Rab3A (data not shown). As with PRA1, the interaction
was abolished when the double Cys prenylation motif of Rab was deleted,
suggesting that prenylation is required for interaction. To confirm
this interaction, we performed an in vitro binding assay
using recombinant Rab1A and Rab3A purified as a 6xHis-tagged fusion
protein from the yeast, Saccharomyces cerevisiae, and
covalently linked to CNBr-activated Sepharose. The proteins were
pre-loaded with GDP, GTP, or maintained in the nucleotide-free state
(in the presence of EDTA). Increasing amounts of recombinant HA-tagged
PRA1 or PRA2 was added to the beads at 4 °C with the bound proteins
recovered and analyzed by Western immunoblot. As shown in Fig.
3A, PRA2 was recovered with the immobilized Rab1A and Rab3A but not with the control Sepahrose beads. There was a slight increase in the amount of PRA2 recovered with
immobilized Rab3A compared with Rab1A. PRA2 showed a slightly higher
affinity for GTP-bound Rab but was clearly recovered with the GDP-bound
as well as guanine nucleotide-free state of both Rab GTPases. Under the
same conditions, PRA1 also showed a slight preference for Rab3A over
Rab1A (Fig. 3B). There was also a slightly higher affinity
for the GTP-bound Rab followed by guanine nucleotide-free and GDP-bound
forms. Thus, we conclude that both PRA1 and PRA2 can interact with, at
least, Rab1A and Rab3A in the guanine nucleotide-bound and free
states.

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Fig. 3.
Binding of recombinant PRA1 and PRA2 to Rab1A
and Rab3A. A, 40 pmol of immobilized Rab1A (top
panel) or Rab3A (bottom panel) was incubated with 10, 15, or 20 pmol of HA-tagged PRA2. The Rab GTPases were preincubated
with GTP, GDP, or EDTA, as indicated. Lane C represents the
control Sepharose beads incubated with 20 pmol of PRA2. B,
the same conditions were used as in panel A except
recombinant HA-tagged PRA1 was added to the immobilized Rab. PRA1 and
PRA2 bound to the beads were detected with anti-HA antibodies.
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PRA2 Is Localized to the ER Compartment--
To determine the
cellular distribution, we subcloned the HA-tagged PRA2 into the
bicistronic expression vector pIRESpuro, transfected it into CHO cells,
and performed a subcellular fractionation analysis. As with PRA1, the
protein fractionated with both the high speed supernatant and membranes
(Fig. 4). When the membrane fraction was
extracted with alkaline carbonate buffer, some of the membrane-bound
protein appeared in the soluble fraction similar to the behavior of
PRA1 and suggested peripheral association with the membrane. However,
the membrane-bound PRA2 partitioned exclusively with the detergent
phase when subjected to Triton X-114 extraction and phase separation.
In contrast, a significant portion of the membrane-bound PRA1 remained
with the aqueous phase in the Triton X-114 extraction. Thus, both PRA1
and PRA2 are highly hydrophobic proteins tightly associated with the
membrane but may also appear in the cytosol.

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Fig. 4.
Subcellular fractionation and extraction of
membrane-bound PRA. Homogenates from CHO transfected with
HA-tagged PRA1 (A) and PRA2 (B) were fractionated
by centrifugation at 100,000 × g for 1 h. The
resulting high speed membrane fractions were further extracted with 0.1 M sodium carbonate, pH 11.5, or with 2% Triton X-114.
S and P refer to the supernatant and pellet
fractions, respectively. Aq and Det refer to the
aqueous and Triton X-114 detergent phases.
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When transfected into CHO cells, PRA2 has a striking reticular staining
pattern reminiscent of the ER (Fig.
5A). Indeed, there was
extensive co-localization with calnexin, a known ER marker. We observed
little, if any, co-localization with mannosidase II, a Golgi membrane
marker. In contrast, PRA1 was found exclusively associated with the
Golgi complex with extensive co-localization with mannosidase II (Fig.
5, C and D). Thus, the two PRA isoforms are
localized to distinct intracellular compartments with PRA2 restricted
to the ER and PRA1 confined to the Golgi complex (30). This distinct
intracellular localization implies that PRA1 and PRA2 may contain
targeting signals that direct the protein to the appropriate
intracellular compartment. It also implies that each PRA might only
interact with a subset of Rab in the cell even though both are capable
of binding multiple Rab isoforms in either the GDP- or GTP-bound
state.

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Fig. 5.
Cellular localization of PRA1 and PRA2.
CHO cells were transfected with HA-tagged PRA2 (panels A and
B) and PRA1 (panels C and D). The
cells were stained with anti-HA (panels A and C),
and with either calnexin (panel B) or mannosidase II
(panel D) to highlight the ER and Golgi complex,
respectively. Scale bar, 2.5 µm.
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The Localization Signal Resides in the Carboxyl-terminal Domain of
PRA--
To define the organelle-specific targeting signal, we
constructed amino-terminal HA-tagged chimeras of PRA1 and PRA2, and determined their cellular localization in transfected CHO cells. Based
on the structural features, we divided the two proteins into three
separate domains: amino-terminal domain (Domain A), the two hydrophobic
segments plus the intervening hydrophilic loop (Domain B), and the
charged COOH-terminal domain (Domain C). Hybrid oligonucleotides
spanning these domains were used to generate the chimeras by PCR using
either HA-tagged PRA1 or PRA2 as the template. As shown in Fig.
6, chimera 1A/2BC, for example, contained
Domain A of PRA1 fused to Domains B and C of PRA2. Likewise, chimera
2A/1B/2C contained Domain A of PRA2 fused to Domain B of PRA1 followed
by Domain C of PRA2, and so on. The various chimeras were subcloned
into the pIRESpuro vector and sequenced to confirm the expected
mutations. Two days after transfection, the CHO cells were stained with
anti-HA and costained with either anti-mannosidase II or anti-calnexin
to identify the Golgi and ER, respectively. As shown in Fig.
7, the COOH-terminal domain of PRA2
targeted the chimera to the ER whether it contained only the
amino-terminal Domain A of PRA1 (1A/2BC in Fig. 7A and
6D), both Domains A and B of PRA1 (1AB/2C in Fig. 7,
B and E), or only Domain B of PRA1 (2A/1B/2C in
Fig. 7, C and F). Similarly, Domain C of PRA1
targeted the chimera to the Golgi complex whether it contained only the amino-terminal Domain A of PRA2 (2A/1BC in Fig. 7, G and
J), both Domains A and B of PRA2 (2AB/1C in Fig. 7,
H and K), or only Domain B of PRA2 (1A/2B/1C in
Fig. 7, I and L). Thus, we concluded that the
COOH-terminal domain of PRA1 and PRA2 contained the localization signal
that either targeted or caused the protein to be retained at the proper
membrane compartment. The amino-terminal Domain A and the two
hydrophobic segments in Domain B of the two proteins were
interchangeable. For PRA1, its normal localization to the Golgi complex
was not altered by the presence of either Domain A (2A/1BC) or Domain B
(1A/2B/1C) of PRA2. Likewise, the normal position of PRA2 at the ER was
not altered by the presence of either the amino-terminal Domain A
(1A/2BC) or Domain B (2A/1B/2C) of PRA1.

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Fig. 6.
Schematic diagram of the PRA1 and PRA2
chimera and their cellular localization. The two proteins were
divided into domains A, B, and C. The PRA1 sequence was represented by
solid figures and PRA2 sequence by shaded
figures. The hydrophobic domains were represented by boxes.
Cellular localization was determined based on co-localization with
calnexin (ER) and mannosidase II (Golgi).
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Fig. 7.
Cellular localization of PRA1 and PRA2
chimeras. The transfected CHO were stained with anti-HA
(panels A, C, E, G, I, and K) and with either
calnexin (panels B, D, and F) or mannosidase II
(panels H, J, and L). The cells were transfected
with chimera 1A/2BC (panels A and B), 1AB/2C
(panels C and D), 2A/1B/2C (panels E
and F), 2A/1BC (panels G and H),
2AB/1C (panels I and J), and 1A/2B/1C
(panels K and L).
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The Carboxyl-terminal Domain of PRA2 Can Partially Target GFP to
the Membrane--
To confirm that the COOH-terminal domain was indeed
responsible for targeting the protein to the appropriate membrane
compartment, we constructed chimeras containing the full-length or only
Domain C of PRA1 and PRA2 fused to the carboxyl terminus of GFP. When transfected into CHO cells, the full-length PRA2 (Fig.
8B) and PRA1 (Fig.
8C) showed a bright fluorescent signal in the ER and Golgi
complex, respectively. Thus, the full-length protein was able to direct
a cytosolic GFP (Fig. 8A) to a specific membrane organelle.
Domain C of PRA2-(132-188) was able to partially target GFP to
intracellular membranes where a distinctive punctate staining pattern
throughout the cell was clearly evident (Fig. 8D). However, membrane targeting by this domain was inefficient as a significant amount of florescent signal remained in the cytosol. Since this domain
of PRA2 contained 57 amino acids, we performed a limited deletion to
further define the boundaries of the localization signal. Further
deletion of 17 residues in GFP-PRA2-(148-188) or 33 residues in
GFP-PRA2-(164-188) completely abrogated this punctate staining pattern
(Fig. 8, F and H). Thus, the minimal localization
signal must either be contained within the first 17 amino acids
immediately COOH-terminal to the second hydrophobic segment or required
domains in addition to that contained within Domain C. In contrast to
PRA2, GFP fusion of Domain C of PRA1-(162-185), which contained only
24 amino acids, cannot target GFP to intracellular membranes (Fig.
8E). Neither can a further addition of 5 hydrophobic residues in GFP-PRA1-(157-185). When combined with the previous observation, these results suggest that the COOH-terminal domain of
PRA2 can partially target proteins to the membrane and that transport
of PRA1 to the Golgi complex required additional domains such as ones
that can functionally interact with Rab or VAMP2.

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Fig. 8.
GFP chimeras of PRA1 and PRA2. The
native GFP showed an even cytosolic pattern in transfected CHO
(panel A) while a GFP fusion with the full-length PRA2
(panel B) and PRA1 (panel C) targeted the protein
to the ER and Golgi, respectively. Representative of the GFP-PRA1
chimeras, GFP-PRA1-(162-185) and GFP-PRA1-(157-185) were shown in
panels E and G, respectively. Representative of
GFP-PRA2-(132-188) was shown in panel D,
GFP-PRA2-(148-188) in panel F, and GFP-PRA2-(164-188) in
panel H.
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Charged Residues at the Carboxy Terminus Direct Organelle-specific
Targeting of PRA--
One striking feature of the COOH-terminal domain
of the PRA isoforms is their overall charge. PRA1 contained a number of
acidic residues whereas the first 15 amino acids of Domain C in PRA2 contained numerous basic residues. The carboxyl terminus of PRA1 contained an additional glutamate immediately after the consensus DXE motif (residues 176-178) involved in the exit of
membrane proteins from the ER (35, 36). To test whether this
DXEE motif might constitute the sorting signal of PRA1, we
mutated these residues to alanine or to basic residues and examined
their effect on cellular localization. Targeting of PRA1 to the Golgi
complex was lost when either one of the acidic residues
(Asp176, Glu178, or Glu179),
both glutamate residues (Glu178 and Glu179),
and all three acidic residues within the DXEE motif were
changed to alanine (Fig. 9A).
It is noteworthy that mutation of the Glu179 residue alone
while leaving an intact DXE motif also abolished localization to the Golgi complex. Mutation of all three acidic residues to lysine also caused mislocalization to the ER. These results
indicate that the DXEE motif is essential for effective Golgi localization of PRA1, and the additional glutamate residue is
indispensable for exit from the ER compartment.

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Fig. 9.
Effect of mutation of PRA1 and PRA2 on
cellular localization. A, mutations introduced to
COOH-terminal residues of PRA1 and their resulting cellular
localization. B, mutations introduced to the COOH-terminal
basic residues of PRA2 and their resulting cellular localization.
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Since the COOH-terminal domain of PRA2 can partially direct GFP to
intracellular membranes and contained a number of basic residues, we
decided to perform a mutagenic analysis of this region. The presence of
basic amino acids immediately following a transmembrane domain on the
cytoplasmic side of cytochrome P-450 has been shown to exclude the
protein from ER transport vesicles (37). However, we observed no
alteration in the ER localization of PRA2 when the basic residues
within the first 15 amino acids (Arg140,
Arg142, Lys145, and Lys147) were
changed to alanine (AAAA in Fig. 9B). Changing the basic residues Arg140, Arg142, Lys145,
Lys147, and Lys151 to glutamate (acidic in Fig.
9B) resulted in partial localization to the Golgi complex.
This might be due to introduction of a di-acidic motif that mimicked a
DXE motif. If so, this suggests that the protein may exit
the ER but is subsequently retrieved. Retrieval of membrane proteins
has been shown to depend on a di-basic KKXX motif located at
the COOH-terminal end (38, 39). Since PRA2 does not contain this
KKXX motif, we explored the possibility that the basic
residues located at the COOH-terminal end, KARE, might mimic this ER
retrieval signal. Mutation of Lys185 or Arg187
to alanine resulted in partial localization to the Golgi complex. Similarly, introduction of either K185A or R187A in the context of a
PRA2 with an acidic COOH-terminal domain resulted in predominantly Golgi localization (acidic/K185A and acidic/R187A in Fig.
9B). Thus, our results indicate that PRA2 can be transported
to the Golgi complex despite the lack of a di-acidic motif for exit
from the ER. But the protein is efficiently retrieved from the
pre-Golgi compartment through a process that partially depended on a
KXRX motif at the extreme carboxyl terminus.
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DISCUSSION |
Proteins involved in vesicle transport are conserved throughout
evolution with specific members of each protein family mediating similar functions at each step of the secretory pathway. We have isolated a second PRA isoform from a rat brain cDNA library based on sequence similarity to a conserved domain in PRA1. Northern analysis
showed that PRA2 is ubiquitously expressed in all tissues, suggesting
that it might also participate in general membrane trafficking events.
Although the overall identity of the two proteins is relatively low, we
concluded that the two proteins belong to the same family based on
their overall structural similarity and ability to bind to Rab. PRA2
also contains two extensive hydrophobic domains (36 and 35 residues).
Subcellular fractionation analysis indicates that the two proteins
share properties of both cytosolic and membrane proteins. A significant
portion of the two proteins fractionated with the high speed
supernatant. The membrane-bound PRA2 behaved as an integral membrane
protein in that it partitioned with the Triton X-114 detergent phase.
However, a fraction of the membrane-bound PRA2 can be extracted with
alkaline carbonate buffer. In the case of PRA1, a portion of the
membrane-bound protein was extracted with alkaline carbonate buffer and
partitioned with the aqueous phase when extracted with Triton X-114.
These properties suggest that PRA might be partially embedded in the
lipid bilayer. A more compelling argument that PRA2 is an isoform of
the PRA family is its ability to bind, at least, Rab1A and Rab3A in an in vitro binding assay. The binding property was very
similar to PRA1 in that both proteins can bind Rab1A and Rab3A in GDP-, GTP-, and guanine nucleotide-free states. This is similar to that of
Mss4, a cytosolic protein that can bind to the transient guanine nucleotide-free state of Rab (40). However, the interaction differs
with respect to the prenylation state of Rab. Binding of Rab to PRA1
and PRA2 is highly dependent on prenylation as deletion of the double
cysteine-motif completely abrogated protein-protein interaction in the
yeast two-hybrid system (not shown). In contrast, Mss4 can interact
with the lipid-unmodified form of Rab (41). Since PRA1 can bind
specifically to VAMP2, we also tested whether PRA2 could interact with
some of the VAMP isoforms. We were unable to detect any interaction
between PRA2 and VAMP1, VAMP2, or VAMP3 (cellubrevin) in the yeast
two-hybrid system and in vitro binding assays (not shown).
Thus, we can exclude VAMP1-3 as possible PRA2-interacting proteins.
Most studies on protein localization within the secretory pathway
focused on sorting and transport of cargo or integral membrane proteins. These proteins enter the secretory pathway by insertion into
or translocation across the ER membrane. The selection thereafter for
transport, retention, or retrieval depends on specific interaction of
localization signals present in the protein with the transport machinery. Similarly, proteins localized entirely on the cytoplasmic side of the membrane contain signals mediating specific targeting to an
organelle. For example, a FYVE finger domain is essential in mediating
specific localization of EEA1 to endosomes (42), a GRIP domain in
targeting to the Golgi complex (43) or a GRASP65-binding site for Golgi
localization of GM130 (26). The two PRA isoforms do not contain any of
these domains, but have very distinct intracellular localization. We
have determined that the sorting signal lies within the carboxyl
terminus following the second hydrophobic domain. This domain clearly
determines the intracellular localization such that replacing the PRA1
COOH-terminal domain with that from PRA2 targeted the chimera to the
ER, the normal cellular localization of PRA2. Likewise, replacing the
PRA2 COOH-terminal domain with that from PRA1 resulted in targeting of
the chimera to the Golgi complex, the normal localization of PRA1. The
importance of this domain was also evident in our previous observation
where its deletion resulted in insertion of PRA1 into the ER membrane
(30). For PRA2, this COOH-terminal domain was able to partially target the cytosolic GFP to the membrane, suggesting that it is capable of
interacting with ER-specific transport machinery. However, efficient
targeting may require an additional domain or perhaps functional
interaction with Rab. This may also apply to PRA1 since the
COOH-terminal domain alone or together with five upstream hydrophobic
amino acids was not able to direct membrane association of GFP.
However, when fused to upstream PRA2 domains, it could target the
protein to the Golgi membrane.
Since PRA shared properties of integral membrane proteins, we examined
whether sorting signals for membrane proteins might be responsible for
PRA-specific targeting. Efficient export of vesicular stomatitis virus
G glycoprotein from the ER requires a di-acidic DXE motif
(35, 36) found on the COOH-terminal side of a YXX
motif
(where
is a hydrophobic residue) at the cytoplasmic side of the
membrane (44). The carboxyl terminus of PRA1 is overall acidic in
nature but contains a DXEE motif (residues 176 to 179).
However, this COOH-terminal domain alone was insufficient in targeting
the cytosolic GFP reporter protein to the Golgi or any intracellular
membrane. In the context of a full-length PRA1, the DXE
motif is essential in Golgi localization and mutation of
Asp176 or Glu178 resulted in retention in the
ER. Interestingly, mutation of Glu179, which contains an
intact DXE motif, also resulted in loss of Golgi
localization. Thus, efficient Golgi localization of PRA1 required an
additional glutamate beside the DXE motif, and our results
suggest that PRA1 might be associated with the transport machinery
utilized by membrane proteins to exit from the ER.
Localization of membrane proteins in the ER may result from exclusion
from ER transport vesicle or retrieval from the pre-Golgi compartment.
In certain cases, a transmembrane domain with basic amino acids
immediately following it on the cytoplasmic side may serve to exclude
the protein from ER transport vesicles (37). We explored the
possibility that the basic residues following the second hydrophobic
domain might mimic this motif thereby allowing PRA2 to remain in the
ER. However, mutagenesis of these basic residues to alanine showed no
effect on PRA2 localization. Retention of membrane proteins in the ER
can also result from protein retrieval from the pre-Golgi compartment,
a process that requires a di-basic KKXX motif at the
cytoplasmic tail (38, 39, 45). PRA2 does not contain a KKXX
motif at the carboxyl terminus, but ends with KARE (residues 185 to
188) that might mimic this retrieval signal. We found that mutation of
either one of the basic residues resulted in partial localization to
the Golgi complex. This suggests that the protein normally transits
from the ER to the Golgi complex, and that mutation of the terminal
basic residues resulted in reduced retrieval from the Golgi complex. It
is interesting to note that PRA2 does not contain a DXE
motif at the COOH-terminal domain, yet is capable of transport to the
Golgi complex. Moreover, transport to the Golgi complex can be enhanced
by the creation of a di-acidic motif at the COOH-terminal domain.
Our data suggest that PRA1 enters the secretory pathway at the ER
compartment and is efficiently transported to the Golgi complex through
its DXEE motif at the carboxyl terminus. It can progress
beyond the Golgi complex since PRA1 was detectable on synaptic vesicle
membrane (46). PRA2 also enters the secretory pathway at the ER
compartment and is transported to the Golgi complex independent of a
DXE motif. It is then retrieved, possibly through its
KXRX motif and returned to the ER compartment.
Thus, intracellular localization of PRA is likely due to its
interaction with the same machinery that sorts proteins for export out
of the ER and retrieval from the pre-Golgi compartment. This might serve to recruit or retain Rab on the transport vesicles. Despite the
broad specificity for Rab, the distinct cellular localization of PRA
implies that each isoform may be restricted in its interaction to a
subset of Rab.