Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305-5307
Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9-GTP with roughly fourfold preference to Rab9-GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (~30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.
RAB GTPases participate in the processes by which
transport vesicles identify and fuse with their cognate target membranes (for reviews see Novick
and Brennwald, 1993 Rabs are doubly geranylgeranylated at their COOH termini, a modification that renders them hydrophobic and is
essential for their activity. While the majority of Rabs are
membrane associated, prenylated Rabs are also found in
the cytosol bound to a protein named GDI (Sasaki et al.,
1990 Rabs, in their active, GTP-bound conformations, are believed to recruit cytosolic factors onto membranes to facilitate subsequent membrane-membrane recognition before fusion (Pfeffer, 1996 We study the Rab9 GTPase and its role in stimulating
the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. We have shown that
Rab9 is required for this process in living cells (Riederer et
al., 1994 Rab9 cDNA clones (Shapiro et al., 1993 Yeast Two Hybrid Screen
Yeast strain Y190 (MATa gal4 gal80 his3 trp1-901 ade2-1-1 ura3-52 leu2-3,-112+URA3::GAL Cloning and Expression
A human Jurkat lymphoma cDNA library (106 plaque-forming units) in
In Vitro Binding Assay
Reactions were in 20 mM Hepes, pH 7.4, 50 mM NaCl, 20 mM imidazole,
5 mM MgCl2, 100 µg/ml BSA. Each prenylated GTPase (1 µg = 571 nM in
complex with BSA or GDI) was incubated with 1 µg (357 nM) recombinant p40 and 100 µM GDP or GTP at 37°C for 1.5 h. p40-bound GTPase
was recovered by copurification on Ni-NTA resin (Qiagen; 20 µl of a 50%
slurry) and elution in 20 µl, 100 mM EDTA; eluted amounts were quantitated by immunoblot analysis. GTPase standards (1-40 ng) were analyzed
in parallel; the amount of GTPase bound to the resin in the absence of p40
( GTPase Assays
Rab9 (50 nM) GTPase activity was measured as described (Shapiro et al.,
1993 Sucrose Gradient Flotation
K562 cell postnuclear supernatant (PNS) was fractionated by sucrose gradient flotation according to Balch et al. (1984). The PNS (6 ml) in 1.4 M
sucrose was overlaid with 3 ml, 1.2 M sucrose and 3 ml, 0.8 M sucrose in an
SW41 tube. Gradients were centrifuged for 3 h at 36,000 rpm. Fractions
(0.5 ml) were collected from the top. Marker protein distributions were
determined by immunoblot after trichloroacetic acid precipitation of 200 µl samples and 12% SDS-PAGE separation.
p40-depleted Cytosol
IgG from preimmune or anti-p40 serum (0.25 ml) was precipitated with
50% ammonium sulfate and pelleted at 95,000 rpm for 10 min in a centrifuge (TLA100; Beckman Instr., Fullerton, CA). Pellets were dissolved in 1 ml K562 cytosol (5 mg/ml) and incubated 5 h at 4°C; protein-A Sepharose
(0.4 ml) was then added for 30 min at 4°C. The slurry was poured into a
column, and the flow through was collected as "depleted cytosol."
We used the yeast two hybrid system (Fields and Song,
1989
A Jurkat cDNA library was screened to obtain a full
length 361 cDNA. Of 6 independent clones identified,
clone 361.6 extended the 5
Clone 361 encodes a hydrophilic protein of 372 amino
acids with a predicted molecular weight of 40,566. We
have termed this protein p40. p40 shares a 50 amino acid
stretch (44% identity) with the Saccharomyces pombe protein, Ral2p (these sequence data are available from GenBank/EMBL/DDBJ index accession number M30827). RAL2 shows genetic interaction with S. pombe RAS1 and
is thought to be involved in the activation of Ras1p (Fukui
et al., 1989 The p40 sequence is comprised almost entirely of six internally repeated sequences of ~50 amino acids in length
(Fig. 3 A). These represent so-called kelch repeats, which
were first detected in the Drosophila kelch protein (Xue
and Cooley, 1993
Purified p40 Binds Rab9-GTP
p40 was expressed in E. coli as an NH2-terminally His-tagged protein and purified to homogeneity (Fig. 4 A, lane
1). Antibodies raised against the recombinant protein recognized a 44-kD band on immunoblots of human cell extracts (Fig. 4 A, lanes 2-4). Fractionation of HeLa cells revealed that p40 is predominantly cytosolic, but a significant
fraction (~30%) was found associated with membranes (Fig. 4 A, lanes 3 and 4).
Fig. 4 B shows that a portion of p40 cofractionates with
Rab9 and MPRs upon sucrose gradient flotation. Endosomes, detected by their content of MPRs (Fig. 4 B, triangles) and the Rab9 GTPase (open circles), fractionated in
two discrete peaks corresponding to the 0.8/1.2 M sucrose
interface and the 1.2/1.4 M sucrose interfaces of this gradient. Soluble proteins remained at the bottom, along with
other dense membrane-bound compartments. Since 70%
of p40 is cytosolic (Fig. 4 A), it was not unexpected that
the majority of the protein was found in the lower half of
the density gradient (fractions 11-19).
To test whether the full length p40 protein interacted directly with Rab9, the proteins were mixed in the presence
of GTP or GDP, and after a period of incubation, p40 and
bound proteins were collected via p40's histidine tag using
Ni-agarose; bound proteins were then identified by immunoblot. As shown in Fig. 5, purified, recombinant p40
bound Rab9-GTP in preference to Rab9-GDP. No Rab9
was detected bound to the resin if p40 was omitted from the reactions (Fig. 5 A).
Rab9 in complex with GDI bound much less p40 than
Rab9-GDP (Fig. 5 B, white bars). Since GDI retains Rab
proteins in their GDP-bound conformations, this result
confirms the lack of interaction of p40 with Rab9-GDP;
GDI could equally well mask a p40 binding site. Consistent with the original two hybrid screen results, p40 bound
very little Rab7 (Fig. 5 B, light gray bars); it also failed to
bind K-Ras (Fig. 5 B, dark gray bars). These experiments demonstrate that the Rab9-interaction domain detected
by the two-hybrid screen is competent for Rab9-GTP association, when present within the structure of the full
length p40 protein. Moreover, the nucleotide-state preference of the interaction was also recapitulated with the full
length protein.
Binding of p40 did not influence the intrinsic nucleotide
exchange rate of Rab9 (Fig. 6 A). In contrast, at micromolar concentrations, p40 inhibited the intrinsic rate of GTP
hydrolysis by Rab9 (Fig. 6 B). While the physiological significance of this observation is not known, this result provides independent confirmation of a direct interaction between p40 and Rab9. Indeed, GTPase inhibition may not
reflect the true role of p40, since Rab9 has a very low intrinsic rate of GTP hydrolysis (Shapiro et al., 1993
p40 Is a Potent Transport Factor
To explore the function of p40, we tested its influence on
an in vitro transport assay that reconstitutes MPR transport from endosomes to the TGN (Goda and Pfeffer,
1988 Our most satisfying result was obtained when we tested
the activity of purified, recombinant p40. As shown in Fig.
7 A, p40 greatly stimulated the overall extent of endosome-to-TGN transport. Under conditions of limiting cytosol to provide other essential transport factors, nanomolar concentrations of p40 enhanced transport to 150% of
the level seen with saturating concentrations of crude cytosolic proteins (Fig. 7 A). Thus, p40 represents a novel
and potent transport factor that stimulates MPR transport.
If p40 functions by virtue of direct interaction with Rab9,
the two proteins might be expected to act synergistically in
transport. In reactions containing limiting cytosol, low concentrations of either p40 or Rab9 (added as an active complex with GDI) alone showed very little stimulation. However,
addition of both of these components stimulated transport
to maximum levels (Fig. 7 B). These data strongly suggest that
p40 functions in concert with Rab9 to facilitate MPR transport.
The experiments described above showed that p40 can
stimulate MPR transport from endosomes to the TGN.
Evidence that the molecule normally functions in this process came from antibody inhibition experiments. Anti-p40
IgG inhibited transport by almost 50% compared with
preimmune IgG (Fig. 8). Furthermore, the inhibition was
neutralized by addition of recombinant p40 (Fig. 8). These
data demonstrate that p40 is normally required for efficient transport. The fact that only partial inhibition was
observed may reflect the fact that our antibodies were
raised against a human antigen while the assay utilizes
CHO cell components.
Membrane Association Accompanies p40 Function
A number of independent lines of evidence support the
conclusion that membrane-associated p40 represents the
active form of the protein. As discussed earlier, ~30% of
p40 is present on membranes. In addition, p40 acts in a
synergistic fashion with presumably membrane-associated
Rab9-GTP. Moreover, recombinant p40 can also bind directly to endosome-enriched membranes (E. Díaz, unpublished observation). Is this the active form of the protein?
While anti-p40 IgG inhibited the overall transport reaction (Fig. 8), cytosol lacking p40 as a consequence of immunodepletion was still fully active in supporting in vitro
transport (Fig. 9), consistent with this possibility. Moreover, preincubation of cytosol alone with anti-p40 IgG did not
inhibit transport in reactions containing untreated membranes (not shown). Together, these data imply that anti-p40
IgG blocks transport by inactivating an active, membrane-associated p40 pool. At minimum, these data show that
membrane-associated p40 is sufficient for MPR transport.
How, then, does soluble, recombinant p40 stimulate transport? It is important to note that p40 stimulated transport
under conditions of limiting cytosolic proteins. We have shown
elsewhere that We have reported here the discovery of a novel transport
factor, p40, that acts together with the active Rab9 GTPase
to drive MPR trafficking from endosomes to the TGN. p40
is present in cytosol and on membranes, a feature common
to a variety of important transport factors including NSF
(Block et al., 1988 How might p40 act? The newly discovered Rab5 effector, Rabaptin 5, has been shown to interact specifically
with the active Rab5 GTPase to drive endosome fusion
(Stenmark et al., 1995 In addition to stimulating endosome-to-TGN transport
at nanomolar concentrations, p40 blocked the intrinsic
GTPase of Rab9, at least when added at micromolar concentrations. We estimate that K562 cytosol contains Other Rab-interacting proteins have been identified
that may also slow GTP hydrolysis. For example, Rabphilin
binds Rab3A-GTP and blocks its GAP-stimulated GTPase
(Kishida et al., 1993 We and others have shown that Rabs are delivered to
distinct membrane-bound compartments in their GDP-bound conformations and only subsequently are these
GTPases converted to their active, GTP-bound forms
(Soldati et al., 1994 Rab GTPase-catalyzed docking interactions will eventually link to SNARE complex formation, before membrane
fusion (Söllner et al., 1993 Vesicles bearing activated Rabs with their recruited
docking factors must then find their targets and undergo
SNARE pairing. In yeast secretion, this would involve the
multisubunit, "Exocyst" complex that contains Sec3p, Sec5p,
Sec6p, Sec8p, and Sec15p (Terbush et al., 1996), which appears to be localized to the bud tip (Terbush et al., 1995).
For homotypic endosome fusion, Rabaptin5 may be sufficient to facilitate v- and t-SNARE pairing. With molecules in hand, the molecular gymnastics accomplished by these
enzymes are now amenable to study.
In summary, we have discovered a highly active transport factor that stimulates the transport of MPRs from endosomes to the TGN. A future challenge will be to determine the subsequent molecular events that are catalyzed
by this novel, Rab-interacting transport factor.
; Zerial and Stenmark, 1993
; Nuoffer
and Balch, 1994
; Pfeffer, 1994
). The strongest evidence suggesting that Rab GTPases function in transport vesicle
docking is the finding that temperature-sensitive mutations in the Sec4p GTPase lead to the accumulation of
secretory vesicles. Rabs are also rate-limiting factors in homotypic membrane fusion events: a GTPase-deficient, activated mutant of Rab5, for example, leads to the generation
of enlarged endosomes (Bucci et al., 1992
), presumably
due to hyperactivation of endosome fusion. Over 30 different Rab proteins have been identified; distinct sets of Rab
proteins are found on the ER, Golgi, intermediate compartment between the ER and Golgi, on early and late endosomes, and on the plasma membrane. Some are redundant
isoforms that carry out a common function (Singer-Krüger
et al., 1994
); most others are unique and essential for a
particular step of intracellular transport.
). We have shown that Rab/GDI complexes represent
active transport factors (Dirac-Svejstrup et al., 1994
) that
possess adequate information to deliver Rab GTPases to
their correct membrane-bound compartments (Soldati et
al., 1994
, 1995
; Ullrich et al., 1994
). After membrane delivery,
Rabs are converted to their active, GTP-bound conformations (Soldati et al., 1994
; Ullrich et al., 1994
), presumably
by the action of a membrane-associated, GDI-displacement factor (Dirac-Svejstrup et al., 1997
) and a distinct, subsequently acting nucleotide-exchange factor (Walch-Solimena et al., 1997
).
). The best characterized example
of this is the recruitment of Rabaptin5 onto Rab5-GTP-bearing endosomes (Stenmark et al., 1995
). Rabaptin5 binds
Rab5 preferentially in its GTP-bound conformation, a process that stimulates subsequent endosome fusion (Stenmark et al., 1995
).
) and in an in vitro system that reconstitutes this
transport process (Lombardi et al., 1993
; Dirac-Svejstrup
et al., 1994
). Given the importance of Rab-interacting proteins in driving vesicle-docking events, we sought to identify proteins with which Rab9 interacts. We report here
the identification of a novel 40-kD protein, p40, that interacts specifically with the Rab9 GTPase in its GTP-bound
conformation and can stimulate the transport of mannose
6-phosphate receptors (MPRs)1 from endosomes to the
TGN in vitro.
Materials and Methods
; Riederer et al., 1994
), anti-Rab9
antibodies (Shapiro et al., 1993
), monoclonal anti-mannose 6-phosphate
receptor antibodies (Lombardi et al., 1993
), prenyl Rab9, bovine brain
GDI, and Rab9/GDI complexes were as described (Dirac-Svejstrup et al.,
1994
; Soldati et al., 1994
). Anti-p40 antiserum was prepared in rabbits using recombinant, His-tagged p40. Endosome-to-TGN transport assays
were carried out by a modification (Itin, C., C. Rancaño, Y. Nakajima,
and S.R. Pfeffer, manuscipt submitted for publication) of the standard
procedure (Goda and Pfeffer, 1988
) using K562 cytosol. p40 and Rab9/
GDI were diluted in cytosol before addition to the assay. Cytosol-dependent transport was determined by subtracting the cpm obtained in the absence of cytosol (~200 cpm). Transport in the presence of 1 mg/ml cytosol was defined as 100%. Rabbit IgG was purified using protein A Sepharose (Sigma Chemical Co., St. Louis, MO). Protein assays were carried out using reagent (Bio Rad; Hercules, CA) and bovine serum albumin as standard.
lacZ, LYS2::GAL
HIS3) was transformed with
the pASI-CYH-Rab9
cc plasmid (Schiestl and Giest, 1989) and colonies
selected by growth on plates lacking tryptophan. Rab9 fusion protein expression was verified by immunoblotting. These cells were then transformed with library DNA (human mature B cell cDNA in pACT) and
plated on synthetic medium lacking tryptophan, leucine, and histidine and
containing 25 mM 3
-aminotriazole (Sigma Chemical Co.). Colonies were
picked after 5 d at 30°C and
-galactosidase activity assessed by colony filter assay (Vojtek et al., 1993
) or quantitative liquid culture assay (Rose
and Botstein, 1983
). Positive clones (His+, LacZ+) were forced to lose the
library plasmid by growth on YPAD containing 10 µg/ml cyclohexamide and were mated to yeast strain Y187 (MAT
gal4 gal80 his3 trp1-901 ade2-101
ura3-52 leu2-3,-112 GAL
lacZ) expressing pASI-CYH-Rab9S21N
cc or -Rab7
cc and tested for
-galactosidase activity. Several clones displayed Rab9-GTP-dependent
-galactosidase activity. Library plasmids
were recovered in Escherichia coli XL1-blue cells and purified on a column (Qiagen, Chatsworth, CA) for subsequent sequencing.
ZAPII (Stratagene, La Jolla, CA) was screened with a 32P-labeled probe
made by random-primed labeling (Boehringer Mannheim, Indianapolis,
IN) of a 400-bp BglII-PvuII fragment representing the most 5
region of
the two hybrid clone. Plasmid DNA was recovered by in vivo excision of
the pBluescript plasmid from the
ZAPII vector in E. coli XL1-blue cells
and purified on a column (Qiagen) for subsequent sequencing. An AvrII-
SalI fragment containing the entire open reading frame of p40 was subcloned into pQE-31 and transformed into E. coli XL1-blue cells. Cells
(OD = 0.9) were induced with 0.5 mM IPTG for 10 h at 22°C. Protein purification was carried out according to the manufacturer (Qiagen), except that cells were broken by two passes through a French press (medium power, 1400 U pressure) and subjected to Sephacryl-S100 gel filtration.
2 ng) was subtracted.
); reactions were analyzed by thin layer chromatography.
Results
) to identify proteins that interact with Rab9 in its active, GTP-bound form. A GAL4 DNA-binding domain
hybrid was constructed using wild-type Rab9 lacking the
two COOH-terminal cysteine residues (Rab9
cc) to avoid
interference due to protein prenylation. To enrich for proteins that interacted specifically with active Rab9-GTP,
we discarded clones that interacted with a mutant of Rab9
(Rab9S21N
cc) that binds GDP with >50-fold preference
to GTP (Riederer et al., 1994
) or a related Rab family
member (Rab7
cc). Two hybrid screening of 1.4 × 106
GAL4 activation domain hybrid transformants led to the
identification of clone 361, which interacted preferentially
with Rab9
cc but not Rab9S21N
cc or Rab7
cc in a
quantitative,
-galactosidase liquid culture assay (Fig. 1).
Clone 361 showed at least fourfold higher
-galactosidase
activity with Rab9
cc than with Rab7
cc (Fig. 1), even
though these proteins are 54% identical (Chavrier et al.,
1990
).
Fig. 1.
Discovery of a
yeast two hybrid cDNA
clone encoding a peptide that
preferentially binds Rab9-
GTP. -galactosidase activity of yeast strains co-expressing the clone 361-GAL4 activation domain hybrid and
GAL4 DNA binding domain hybrids of either Rab9
(black), Rab9S21N (white),
or Rab7 (gray).
[View Larger Version of this Image (23K GIF file)]
region of the two hybrid clone
361 by 589 bp (Fig. 2) and contained a potential ATG start
codon (Kozak, 1987
). The 5
region of this clone was virtually identical to a human EST (these sequence data are
available from GenBank/EMBL/DDBT under accession
number N36641). Furthermore, two in-frame stop codons
were found upstream of the potential initiator codon (Fig.
2, underlined). Thus, clone 361.6 represents a full length
cDNA.
Fig. 2.
cDNA and predicted amino acid sequence of p40. The
1,298-bp cDNA and predicted 372 amino acid protein are shown.
The two in frame stop codons are underlined. The portion of the
cDNA identified in the two hybrid screen began at base pair 564.
[View Larger Version of this Image (50K GIF file)]
); thus, p40 contains a domain in common with
another small GTPase activator.
) and are found in a wide variety of proteins of completely unrelated function (Bork and Doolittle,
1994
). Kelch repeats are predicted to form four-stranded,
anti-parallel
sheets that assemble into propeller-like barrel structures. The repeat is characterized by a pair of glycine residues at positions 15 and 16, immediately preceded
by two hydrophobic amino acids, a tyrosine, and a fourth
hydrophobic residue (Bork and Doolittle, 1994
). In p40,
phenylalanine is found at position minus one relative to
the glycine pair in four of six of the repeats, and valine or
isoleucine is always present at position minus two. However, only two of the p40 kelch repeats contain the upstream tyrosine residue. A tryptophan residue (found
in other kelch motif-containing proteins) and a proline
(unique in p40) are found in all of the repeats at position
+20 and +30, respectively, downstream of the glycine
pair. Due to the kelch repeats, the entire p40 structure can
be predicted as drawn in Fig. 3 B. In this representation, the sequences homologous to S. pombe Ral2p are shown
in bold; they fit readily into a connected pair of
strands
that may comprise a portion of the Rab9-interacting region. Circular dichroism analysis of p40 failed to reveal the
presence of any predominant
helices, consistent with the
structure shown (data not shown). In kelch proteins of
known tertiary structure, enzyme active sites are created
by the loops located at the top of the barrel structure (Bork and Doolittle, 1994
). Thus, p40 is predicted to fold
into a compact
barrel that may interact with Rab9 via
the
sheet connecting loops.
Fig. 3.
(A) Alignment of kelch repeats in p40. h, Hydrophobic
residues; t, turns. Bold residues indicate conservation with the
original kelch motif. Also highlighted in bold in p40 are proline
residues, whose positions are conserved among p40 repeats but
are not present in other kelch-motif proteins. (B) Schematic diagram of the postulated barrel structure of p40 showing the region of Ral2p similarity (bold) and a large loop domain. The
dashed line does not represent additional amino acid sequences.
The portion of the protein shown to interact with Rab9 by two
hybrid analysis is indicated at the bottom.
[View Larger Version of this Image (39K GIF file)]
Fig. 4.
Subcellular distribution of p40. (A) Coomassie blue-stained SDS-PAGE of purified, recombinant, His-tagged p40 and
immunoblot of cell extracts. Lane 1, recombinant p40 (1 µg); lane
2, K562 cytosol (100 µg); lanes 3 and 4, HeLa cytosol or membranes (each ~100 µg, or 10% of a 10-cm dish). (B) A portion of
p40 cofractionates with Rab9 and mannose 6-phosphate receptors upon sucrose gradient flotation of K562 cell postnuclear supernatant. The top of the gradient is at the left.
[View Larger Version of this Image (29K GIF file)]
Fig. 5.
Purified p40 binds Rab9-GTP in strong preference to
Rab9-GDP. (A) An example of immunoblot binding data obtained is shown. Values shown in B were determined by PhosphorImager quantitation; Rab9 standards were included on the
gels to permit determination of the nanogram amounts of Rab9
bound.
[View Larger Version of this Image (32K GIF file)]
). Together, these data demonstrate that p40 interacts directly
with Rab9-GTP.
Fig. 6.
p40 inhibits Rab9's GTPase activity but not its rate of
nucleotide exchange. (A) Nucleotide exchange was assayed with
10 nM Rab9 and 100 nM p40 according to Soldati et al. (1994).
(B) Effect of p40 on the Rab9 GTPase. Reactions contained 50 nM Rab9. Control reactions displayed a rate of 0.0122 min
1, in
agreement with previously reported values (Shapiro et al., 1993
).
[View Larger Version of this Image (15K GIF file)]
). Transport requires Rab9 (Lombardi et al., 1993
;
Dirac-Svejstrup et al., 1994
), NSF, and
SNAP (Itin, C.,
C. Racaño, Y. Nakajima, and S.R. Pfeffer, manuscript submitted for publication) and shows all of the biochemical characteristics unique to this transport process (Goda and
Pfeffer, 1988
).
Fig. 7.
p40 functions in endosome-to-TGN transport. (A) Recombinant p40 was added to reactions containing 0.6 mg/ml cytosol. No additional stimulation was observed at higher p40 concentrations. (B) p40 and Rab9/GDI act synergistically in MPR
transport. Recombinant p40 (10 ng/ml), Rab9 in an equimolar
complex with GDI (100 ng/ml), or both components were added
to transport reactions containing 0.65 mg/ml cytosol. Transport is
presented in relation to that seen with 1 mg/ml cytosol (2,345 cpm). Error bars represent standard error of the mean (n = 2).
[View Larger Version of this Image (26K GIF file)]
Fig. 8.
Anti-p40 antibodies inhibit MPR transport.
Immune or preimmune IgG
(75 µg/ml) was added to
MPR transport reactions
containing 1 mg/ml cytosol
and where indicated, p40
(100 ng/ml). Error bars represent standard error of the
mean (n = 2). Control reactions yielded 724 cpm.
[View Larger Version of this Image (76K GIF file)]
Fig. 9.
Depletion of cytosolic p40 does not inhibit
MPR transport. (A) Anti-p40
immunoblot analysis of cytosols (100 µg each) preadsorbed with either preimmune IgG (control) or anti-p40 immune IgG (depleted). (B) Transport activity of the
cytosols shown in A. Cytosols
were assayed at 0.4 mg/ml;
100% transport of control cytosol represents 556 cpm.
Values shown represent the
average of duplicate determinations (standard error 5%) in a representative experiment.
[View Larger Version of this Image (29K GIF file)]
SNAP is among the most limiting cytosolic
factors under these conditions (Itin C., C. Racaño, Y. Nakajima, and S.R. Pfeffer, manuscript submitted for publication). Presumably, by driving more p40 onto membranes, the reaction can bypass limitations of other factors.
Further experiments will be needed to identify directly the
proteins to which p40 binds on membranes.
Discussion
),
SNAP (Clary and Rothman, 1990
),
p115 (Waters et al., 1992
), and Rabaptin 5 (Stenmark et
al., 1995
). An important characteristic of p40 is its specific interaction with the active conformation of the Rab9 GTPase. Since Rabs likely function in the process of vesicle
docking, we infer that p40 also functions at this stage of vesicular transport.
). Rabaptin 5 is part of a much larger
multiprotein complex. This feature led us to propose that
GTP-bearing Rabs function by recruiting large, coiled-coil, macromolecular docking factors to the surface of
transport vesicles to facilitate the docking reaction (Pfeffer, 1996
). However, unlike Rabaptin 5, p40 occurs as a
monomer in cytosol (as determined by gel filtration of
K562 cytosol), and thus it might represent a different class
of Rab-interacting transport factor. Alternatively, p40
may be part of a larger complex when present on membranes. Indeed, p40 could act to catalyze the assembly of
other docking factors on the surface of transport vesicles carrying MPRs from late endosomes to the TGN. It will be
of interest to characterize further the interaction of p40
with membrane-associated components.
0.05
µg/mg cytosol protein, or ~50 nM p40. Unless there are
significant, local concentration effects on the surface of
membranes, it does not seem likely that p40 acts to block
Rab9 GTPase in vivo, since it is present in insufficient levels to do so. It is quite possible, however, that local membrane concentration effects are significant. In any event, it
is essential to note that the ability of a protein to block
GTPase activity may simply be an indirect consequence of
tight binding to the GTP-bound form. A secondary outcome of this interaction would be to retain the Rab in its
active conformation. Whether or not p40 blocks Rab9 GTPase, it is satisfying to note that a minimal estimate for
membrane-associated Rab9 is also ~50 nM, a value quite
close to the level of p40.
). At the nerve terminal where vesicles
may wait days before exocytosis, such a factor could be important to retain synaptic vesicles in an exocytosis-competent state. Whether this feature is also critical for a constitutive process such as endosome-to-TGN transport is not
yet at all clear.
; Ullrich et al., 1994
). Walch-Solimena
et al. (1997)
have now reported that Sec2p has the capacity
to stimulate nucleotide exchange on Sec4p, converting it
to the GTP-bound, active form. Satisfyingly, Sec2p interacts directly with the vesicle-associated, Sec4 GTPase and
is necessary for subsequent secretory vesicle localization
to the bud tips in yeast (Walch-Solimena et al., 1997
). If
this paradigm holds true for other transport steps, it would
seem that Sec2p-like nucleotide exchange factors would
act before p40 or Rabaptin, to convert the respective GTPase to its active form. It has been postulated that proteins
related in sequence to Sec2p, of which there are several,
including the Rab3A-interacting, Rabin 3 protein (Brondyk
et al., 1995
), activate Rabs once delivered to cellular membranes (Walch-Solimena et al., 1997
). The action of such
proteins would then lead to the recruitment of docking
factors onto the vesicle surface. Not yet known is whether
Sec2p-like proteins remain bound to the GTPase after
converting the proteins to their active conformations.
; Lian et al., 1994
; Rothman,
1994
; Søgaard et al., 1994
; Pfeffer, 1996
). In this regard it is
interesting to consider the need for 30 distinct Rab GTPases. While v- and t-SNAREs are likely to encode specific,
cognate, vesicle-targeting information, genetic studies in
yeast suggest that pairing of v- and t-SNAREs requires additional and distinct sets of both docking factors and Rab
GTPases. Some of the functions of these distinct components may be to activate distinct t-SNAREs, perhaps by
releasing SNARE-protecting proteins (Pfeffer, 1996
). Thus,
some of the specificity of vesicle docking is indirectly provided by Rab GTPases.
Received for publication 30 April 1997 and in revised form 29 May 1997.
This research was funded by a research grant to S.R. Pfeffer from the National Institutes of Health (DK37332), a predoctoral fellowship from the National Science Foundation to E. Díaz, and by a postdoctoral fellowship from the Human Frontier Science Program Organization and CIBA-Geigy Jubiläumsstiftung to F. Schimmöller.We are grateful to R. Lesley, B. Oh, and W. Choi for their assistance, C. Itin for help with the transport assay, D. Laurents for CD analysis, S. Elledge for the yeast strains and plasmids, W. Wang and G. Crabtree for the Jurkat cDNA library, and M. Garrett for purified K-Ras and anti-K-Ras antibodies.
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