Download REVIEWS Structural insights into the function of the

REVIEWS
TIBS 21 - DECEMBER 1996
Structural insights into the function
of the Rab GDI superfamily
Shih-Kwang Wu, Ke Zeng, lan A. Wilson
and William E. Balch
The 1.81~, crystal structure of Rab GDP-dissociation inhibitor (GDI), a
protein that plays a critical role in the recycling of Rab GTPases involved
in membrane vesicular transport, has been recently determined.
Biochemical studies implicate a highly conserved region involved in Rab
binding, which is common to both GDI and the evolutionarily-related
choroideremia gene product (CHM/REP) required for Rab prenylation.
Here, we summarize the mechanisms by which members of the GDI superfamily might function to coordinate events leading to membrane fusion,
and we discuss the unexpected, yet striking structural homology of GDI to
FAD-binding proteins.
MOVF_JVlF.J~ITOF PROTEINS along the
exocytic and endoc~ic pathways of the
eukaryotic cell occurs by small vesicular carriers that mediate selective
transport 1. Integral to this process are
a large group of prenylated small
GTPases encoded by the rab gene family. Rab proteins exist in both inactive
GDP-bound and active GTP-bound forms.
Conversion between the two forms allows Rab to function as a molecular
switch to control the assembly and disassembly of protein complexes involved
in membrane fusion 2. Rab function is, in
turn, promoted by factors that either
accelerate guanine-nucleotide exchange
(GEO, stimulate guanine-nucleotide hydrolysis (GAP) or prevent nucleotide
dissociation (GDI)3. GDI is a particularly
intriguing protein in that it appears
to recycle Rab GTPases in a fashion
analogous to the role of [3~/subunits of
heterotrimeric (~[3~/) G proteins by promoting the release and retrieval of the
~-GTPase4. GDI is now recognized to be
a member of a larger group of GDI-like
proteins, which includes the choroideremia gene product CHM [also referred to as Rab-escort protein (REP)]
S-K. Wu is at the Department of Cell Biology;
K. Zeng is at the Departments of Cell and
Molecular Biology; I. A. Wilson is at the
Department of Molecular Biology and the
Skaggs Institute for Chemical Biology; and
W. E. Balch is at the Departments of Cell and
Molecular Biology, The Scripps Research
Institute, 10550 N. TorreyPines Road,
La Jolla, CA 92037, USA.
472
involved in the geranylgeranylation of
Rab proteins 5. Given the central roles of
GDI and CHM/REP in membrane vesicular traffic, we highlight evidence that
identifies the function of amino acids
conserved among members of the GDI
superfamily in Rab recognition and recycling. In addition, we explore the surprising similarity of GDI to flavoproteins,
which suggests that GDI might have
an as yet unanticipated activity in living cells.
GDI functions as a recycling factor
Why is GDI important? Vesicular traffic requires the continuous recycling
of components that dictate sequential
events leading to membrane fusion between carrier vesicles and subcellular
organelles. Some factors are integral
membrane proteins and are recycled
by vesicle-mediated mechanisms, while
others reside only transiently on the
membrane. Of these, some are soluble
and can be recycled by simple diffusion
through the cytoplasm. Others, such as
Rab proteins, are peripheral membrane
proteins that are hydrophobic in character owing to the post-translational
addition of geranylgeranyl lipids to
their carboxyl termini; therefore, these
require an 'escort' service to mediate
membrane attachment and retrieval.
GDI provides this service ~ig. 1). GDI
and the GDP-bound form of Rab are
present in the c~oplasm as a soluble
~80kDa complex that delivers Rab to
the membrane in response to unknown
9 1996, Elsevier Science Ltd
signaling events. In the subsequent step
of the pathway ~ig. 1), GDI is released
to the c~osol and Rab remains membrane-associated, where it is activated to
the GTP-bound form by GEE Following
membrane fusion and inactivation by
GAP, GDI extracts the GDP-bound Rab
from membranes to the c~osolic reservoir for re-use ~ig. 1).
GDIisoforms
The ~-isoform of GDI has been
mapped to the gene rich G6PD locus in
human Xq28 ~ef. 6). The amino acid
sequence of GDI is well conserved from
yeast to man (>50%), with >80% identity
between mammalian homologues 7. In
mammals, there are three recognized
isoforms, although Southern blot analysis of genomic DNA suggests that up to
five isoforms are present in mouse and
rat 8. Of the two major isoforms (~ and 13)
characterized to date from mammalian
cells, the c~-isoform is largely confined
to brain tissues whereas the [3-isoform
is ubiquitously distributed 7,9. Interestingly, all GDI isoforms appear to bind a
similar spectrum of Rab proteins in vitro.
The relative tissue abundance of different isoforms is therefore an important
factor in dictating which population of
Rabs is associated with a particular
GDI isoform. However, at least one isoform, the mouse GDI-2 (the ~/-isoform)
is specifically enriched in endosomal
compartments ]~ suggesting a more specialized function in the endoc~ic recycling pathway. The residues that are
responsible for potential differences in
function of the a-, 13- and ~-isoforms remain to be elucidated. In yeast, only a
single gene (GDII) has been detected
and its protein product is essential for
cell growth 11. While GDIs that recognize
the Pdao-related small GTPases have
been characterized 12, they show no
sequence or functional overlap with
Rab-GDI.
GDI binds Rab in the GDP-boundform
~-GDI was first isolated from bovine
brain based on its ability to inhibit the
intrinsic dissociation of GDP from the
Rab3A protein, a GTPase !nvolved in
vesicle fusion at the synapse 13. While
Mg2+is critical for the stable association
of both GDP and GTP to Rab proteins,
GDI markedly reduces the intrinsic dissociation of GDP, but not GTP. The ability to bind and stabilize Rab in the
GDP-bound form is a general functional
property of GDI7. Consistent with a role
in Rab protein recycling, different c~osolic GDI-Rab complexes have been
PII: S096&0004(96)10062-1
REVIEWS
TIBS 21 - DECEMBER 1996
shown to be essential for transport
between both exocytic and endocytic
compartments in vitro 14-16.The ~-isoform
of GDI found in the cytosolic complex
with Rab5 has been shown to be
phosphorylated, whereas a transient,
membrane-associated form is not 17.
Such results raise the distinct possibility that phosphorylation might serve
as a reversible modification controlling
GDI function.
The mechanism by which Rab proteins are delivered to or extracted from
membranes by GDI remains to be
resolved ~s,~8(Fig. 1). Recruitment of the
GDI-Rab complex to the membrane is
likely to be triggered by components involved in the assembly and maturation
of transport vesicles or the priming of
organelles for fusion. In each case, Rab
serves as the specificity module for
delivery to a particular compartment.
Thus, regions involved in Rab binding
and receptor recognition would be predicted to be functionally linked. Binding
to membranes might involve a specific
receptor, which is itself a Rab GEE leading to Rab activation. Alternatively, it
might involve two separate proteins,
the first required to promote membrane
association of the GDI-Rab complex, the
second being a Rab GEE which promotes
Rab activation and GD1 release ~5,~8.The
latter is an attractive model given the
notable lag period between the membrane association of the GDI-Rab complex and guanine-nucleotide exchange
on Rab. Following membrane fusion,
specific extraction of Rab might occur
in response to being discharged from
components of the transport machinery
in the GDP-bound form. Alternatively, it
might require that Rab (GDP-bound) is
associated with a new factor(s). That a
single GDI species can function at multiple transport steps and with multiple
Rab species is evident from an analysis of GDII function in yeast. Depletion
leads to inhibition of transport in both
the exocytic and endocytic pathways u.
GDI and the choroideremiagene product
form a superfamilyof GDI4ikeproteins
Rab-GD] is significantly (~30% identity) related to the mammalian choroid-
eremia gene product chm. Mutations in
the X-linked chm gene contribute to
late-onset retinal degeneration and loss
of vision ~9. CHM is identical to the component A subunit REP1 of Rab geranylgeranyltransferase II, a hetero-oligomeric
protein complex that is responsible for
prenylation of Rab proteins terminating
in CxC (x is any amino acid) or CC 20,21.
Donor membrane
....
GTP
3
GTP
GDP
GTP
D~,
\
~
......~i!~
....
Target membrane
Figure 2.
The GDP-dissociation inhibitor (GDI)-Rab cycle. The GDP-bound form of Rab exists exclusively as a soluble complex with GDI in the cytoplasm. This complex serves as a reservoir
to deliver Rab to the membrane during assembly of a transport vesicle or the priming of an
organelle for fusion. During or following dissociation from GDI, Rab is activated to the GTPbound form by a guanine-nucleotide-exchange factor (GEF), which subsequently participates in vesicle docking and fusion. Following GTP hydrolysis, promoted by guaninenucleotide-activating protein (GAP), Rab is returned to the GDP-bound form, where it is
extracted by GDI for subsequent reuse.
Two mouse isoforms [REP1 and REP2
(also referred to as CHML)] with 72%
identity have been characterized.
Members of the CHM/REP family escort
newly synthesized Rab proteins to the
catalytic subunits of the geranylgeranyltransferase complex for prenylation 22.
Like GDI, REP only binds the GDP-bound
form of Rab23. Biochemical studies have
detected only limited differences in Rab
substrate specificity for the two different isoforms. In choroideremia lymphoblastoid cells deficient in REP1, nearly
all Rab proteins are prenylated by
REP2; only one novel isoform, Rab27,
was found in the unprenylated state z4.
As this Rab is present in both retinal
and choroidal epithelial layers that degenerate in choroideremia, the defect in
Rab27 prenylation is believed to be the
underlying basis for disease. Although
GDI cannot substitute for CHM/REP in
geranylgeranylation, REP1 has been
shown to function in an identical fashion to GDI in delivering prenylated Rab
to membranes 2s. Indeed, the delivery of
newly synthesized Rab to membranes
by REP appears to be a prerequisite for
interaction with GD126. The CHM/REP
homologue in yeast, MRS6p (MS14p),
is an essential gene and has similar
biochemical properties to mammalian
CHM/REP in geranylgeranylation of yeast
Rab homologues 27,28.
Alignment of GDI and CHM/REP
family members shows regions of primary sequence which are highly conserved (referred to as Sequence Conserved Regions or SCRs) (Fig. 2a).
Although a variable insert of differing
length separates SCR1 from SCR2 in
GDI and CHM/REP (Fig. 2a), regions of
identity can be detected throughout
the entire sequence 4. Residues comprising motifs found in SCRs 1A, 1B and
3B are particularly diagnostic of GDI
and CHM/REP family members owing
to their composition of invariant diand tripeptides ~9. These particular
motifs, defined by the consensus sequences D6VxxxGTGxxExxL (SCR1A),
G26xxVLHxDxxxYYxGxxY (SCR1B), and
G232ExxQGFxRxxAxxG (SCR3B) (~-GDI
numbering 29) are common to members
of what might now be referred to as a
GDI superfamily.
473
REVIEWS
TIBS 21 - DECEMBER 1996
(a)
SCR
1A 1B
SCR
2
SCR
3A 3B
Bovine GDI
]F-]FI
Human GDI
]FIE]
Drosophila GDI
]FIE]
Yeast GDI
]FIE]
Yeast
MRS6/MS14
Human CHML/REP2
]
~!
Human CHM/REP1
]
]
~'~ ;~j ~,
patient point mutations
I
0
I
I
1O0
I
I
200
I
I
f
I
300
400
Amino acid residues
I
I
500
I
I
600
Figure 2
(a) Conserved domains among GDP-dissociation inhibitor (GDI) and CHM/REP family members. The
schematic diagram (modified from Ref. 4) illustrates the location of sequence conserved regions
(SCRs), which contain consensus motifs common between members of the GDI superfamily. The arrows indicate locations of point mutations in human CHM/REP1 that lead to disease. (b) The structure of GDI reveals conserved and non-conserved faces and a Rab-binding region (GCD) located at the
apex of GDI. The locations of SCRs 1-3 are color-coded as indicated in (a): SCR1, yellow; SCR2, red;
SCR3A, brown; and SCR3B, orange9 The insert region corresponding to residues connecting SCR1
and SCR2 is shown in blue. The structure in the left panel has been rotated 90 ~ about the vertical
axis relative to the right panel. Figure modified from Ref. 4.
the solution of the crystal
structure of the GDI-Rab complex. However, some insight
can be gained from the structure of the H-ras-related
GTPase, RaplA, in complex
with the Ras-binding domain
(RBD) of cRafl kinase, cRafl
kinase functions as a GDI for
GTP-bound forms of Ras-like
proteins 3~ Complex formation
occurs through an extended
B-sheet involving the effector
domain of RaplA3~ The conformation of the effector domain
of Ras-superfamily GTPases is
particularly sensitive to the
GDP- or GTP-bound state of
the protein and functions as
a molecular switch allowing
these small GTPases to interact with different upstream and
downstream components.
We4 and others 26,31 have
found that amino acid residues in the [32/loop 2 region of
the Rab effector domain are
also essential for binding GDI.
By analogy to the RaplA-RBD
complex, Rab might dock to
GDI through the B-strand of its
effector loop by an analogous
extended B-sheet configuration
(Fig. 3a), although the B-sheet
folds that provide a framework for such interactions are
inherently different in RBD and
GDI. This orientation would
place the ~2/L5 and a3/L7
regions, previously implicated
in Rab function 32,33, at the
interface with GDI (Fig. 3a). As
this interaction might accommodate only one conformational state of the effector
domain, it would explain why
GDI might only detect GDPbound forms of Rab.
SCRs I and 3B form a compact Rab-binding the GDI-CHM consensus Domain (GCD) Similar topologicalorganizationsof GDI and
(Fig. 2b, yellow and orange) 4. The highly CHM/REP
region
The X-ray structure of c(-GDIrevealed conserved residues Tyr39, Glu233 and
that all of the SCRs are found on one Arg240 in SCRs 1B and 3B are found in
face of GDI4 (Fig. 2b). This conserved surface-exposed strands and helices
face is therefore likely to play a critical forming the GCD with their sidechains
role in the interaction of GDI and 9pointing into a cleft in the upper half of
CHM/REP with Rab and/or membrane- GDI4. When these and adjacent residues
associated receptors involved in Rab re- are mutated, GDI loses its ability to bind
cycling. Examination of the structure of Rab4. As GCD is highly conserved evoGDI also revealed that SCR1 (Fig. 2b, lutionarily, it might be diagnostic of the
yellow) and SCR3B (Fig. 2b, orange), a general Rab-binding region in all GDI
motif separated from SCR1 by nearly superfamily members.
How does Rab dock to the GCD? The di140 residues, fold to form a compact region at the apex of GDI referred to as rect answer to this question will require
474
The structure of GDI has important
implications for the structure and function of CHM/REE Sequence alignment
suggests that the overall topology of
members of the CHM/REP family is
likely to be very similar to that of a-GDI4.
The only major apparent structural difference is the presence of a larger insert between SCR1 and SCR2 in yeast
MRS6 and in human/mouse CHM/CHML
than in GDI (Fig. 2b, blue). This insert
in CHM/REP might provide additional
flexibility to bind both prenylated and
II
TIBS 2 1 - DECEMBER 1996
REVIEWS
non-prenylated forms of Rab,
(b)
and/or be involved in the
f"
recognition of the catalytic
subunits of geranylgeranyltransferase I1.
The potential role of the
effector domain of Rab in
CHM/REP recognition is controversial: mutational analyses
of different Rab proteins have
yielded conflicting results when
comparing in vitro binding
assays to in vivo functional
analyses 31,34,3s. Recent studies
suggest that at least one effectot domain residue that is
essential for recognition of
GDI is not required for prenylation 26. However, a number of
lines of evidence argue that
the effector domain might be
required: REP1 recognizes only
the GDP-bound form of Rab23,
simple peptidomimetics to the
carboxyl termini of Rab family
Figure 3
members are not substrates for
(a) A potential model for docking of Rab with GDP-dissociation inhibitor (GDI). The structure 45 of the
geranylgeranyl transferase II
Rab-related H-ras protein (GDP-bound form) (grey) was used to simulate the docking of Rab to GDI
(Ref. 36), and truncated Rab
(green). An extended 13-sheetis illustrated by the B-strands present in H-ras (purple) and the 13-strands
proteins that lack terminal
in e-GDI (orange), which form part of the GDI-CHM consensus domain (GCD) region. The coordinates
CxC or CC acceptor residues
of H-ras (PDB 4q21) were taken from the Brookhaven databank. (b) The location of residues involved
efficiently inhibit prenylation
in FAD binding to flavoproteins. The structure is rotated ~180 ~ about the vertical relative to (a) to
of wild-type Rab2~ Further exbring the FAD-binding groove and the ~(~13unit (yellow) and the remnant GXG motif (red) into view. The
orange strands flanking the 13(~i3unit are part of the extended sheet projected to be involved in Rab
periments will be necessary
binding shown in (a). The ball-and-stick figures show the projected location of FAD (large molecule octo clarify this issue.
cupying groove) based on superimposition of PBHase with GDI.
The structure of GDI provides important insight into
genetic defects leading to choroideremia. discovery. Both the three-dimensional projected location of FAD in GDI based
The mutation spectrum in Danish and folding and topological connections of on superimposition with PBHase illusSwedish chm genes includes both de- a-helices and B-strands are strikingly trated in Fig. 3b is found to occupy a
letions and point mutations, which give similar to previously characterized flavo- similar groove to that found in flavorise to premature stop codons, leading proteins, including p-hydroxybenzoate proteins. Despite this strong structural
to a truncated protein 19(Fig. 2a). Such hydroxylase (PBHase), cholesterol oxi- conservation, FAD was not found in the
truncations are presently confined to dase (COX), glucose oxidase (GOX) corresponding region of the GDI strucresidues found in the carboxy-terminal and glutathione reductase (GR)4. These ture prepared from recombinant or nahalf of CHM/REP1, downstream of SCR3B four proteins are FAD-dependent flavo- tive sources 4 (K. Zeng, W. E. Balch and
(Fig. 2a). Deletion of 70 carboxy-terminal proteins that have little sequence simi- I. Wilson, unpublished). As nearly 70%
amino acids leads to loss of CHM/REP1 larity between one another or with GDI. of the structure of oL-GDIcorresponds
function in vivo and in vitro 19. The In these flavoproteins, the ADP moiety to that of a flavoprotein, this raises
molecular explanation for this is readily of FAD is bound to a conserved amino- previously unsuspected possibilities reapparent in the structure of GDI. terminal structural unit consisting of garding GDI and CHM/REP function in
Deletion of even a few residues at the a sequential 13-strand, an a-helix and a the cell.
carboxyl terminus would be predicted [3-strand (referred to as the 13al3 unit).
Flavoproteins catalyse a broad specto disrupt the tight hydrophobic pack- This [3~[3 unit contains a consensus trum of redox processes involving
ing underlying the structural organiz- GxGxxG sequence (x represents non- molecular oxygen. The proteins most
ation of GCD4 and either lead to com- glycine residues) involved in phosphate structurally related to GDI (PBHase,
plete loss of activity, and/or target the binding in many nucleotide-binding pro- COX, GOX and GR) can be generally
misfolded protein for degradation. In teins 37. The tightly bound FAD in flavo- classified into three groups: (1) oxithe case of CHM/REP, these would lead proteins occupies a long groove extend- dases that generate oxidized flavin
to disease.
ing between the 13u[3 region and the and H202 as the end products (GOX);
base of the protein s8-4~
(2) mono-oxygenases (hydroxylases),
Structural similarity between GDI and
Intriguingly, the 13c~13region is found which induce splitting of the O-O bond
flavoproteins
in a similar structural location in GDI by inserting one of the oxygen atoms
A search for proteins structurally (Fig. 3b, yellow) along with a residual into the substrate [such as p-hydroxysimilar to GDI led to a surprising GxG motif (Fig. 3b, red). Indeed, the benzoate (PBHase) or cholesterol (COX)]
475
REVIEWS
TIBS 2 1 - DECEMBER 1996
References
D
and reducing the other to H20; and (3)
proteins that have auxiliary redox centers linked to the bound flavin (GR)4L
As yet, there is no known requirement
for FAD or other nucleotides in the function of GDI or CHM/REP. One possibility
is that GDI is derived from a common
ancestral fla~n-binding protein that is
now functionally independent of the cofactor. If so, then why did Rab binding
evolve from proteins mediating redox
processes?
Am alternative interpretation is that
the role of FAD in GDI function is transient and differs from standard flavoproteins. In support of the latter possibility, GDI lacks basic residues typically
involved in neutralizing the phosphate
groups in flavoproteins containing
tightly bound FAD. The association of
FAD with GDI could be similar, in principle, to that in NADPHoxidase, a multisubunit protein complex involved in
the FAD~ependent oxidative killing in
the phagosome of neutrophils. The
gp91-phox or c~ochrome bss8 subunit binds FAD very weakly and is
lost during purification 42. Interestingly,
cytochrome bs58 binds to and is regulated by RaplA43,44. Whether GDI also
functions in a Ra~regulated redox
event represents an intriguing possibility. In addition, M~6p/MS14p [the
yeast homologue to CHM/REP (Fig. 2a)]
was first identified as a suppressor of a
mutant that prevents the function of the
mitochrondrial respiratory cycle29.
Rab proteins to different subcellular
compartments? Given the diversity of
Rab proteins and the specificity of
GDI-CHM/REP for GDP-bound forms,
residues common to switch regions of
different Rabs are likely to be at least
part of the answer. In GDI, less conserved residues might augment the
specificity of various isoforms for different Rabs. A second area requiring attention will be to understand the molecular basis for delivery and extraction
of Rab from membranes. While the conserved face of GDI is an attractive region, it might be that residues found
adjacent to GCD function in this capacity to link Rab displacement to local
changes in conformation. Finally, beyond the ob~ous need to understand
the potential role of FAD in GDI function, the structural relatedness of the
GCD region of GDI to that found in
PBHase, COX, GOX and GR raises the
possibility that mammalian flavoproteins might be subject to regulation
by Rab or other small GTPases. The
elucidation of the structure of GDI
has already provided tangible insights
into its role in Rab recycling and stimulated exciting new lines of investigation regarding the regulation of GDI
and CHM/REP function in vivo. Future
structural studies should help to illuminate additional mechanistic properties
of this key protein in membrane vesicular traffic.
Acknowledgments
Future dite~'tions
Given the central function of GDI and
CHM/REP in Rab protein prenylation
and recycling, a large number of questions remain to be explored. How do
these proteins recognize and mediate
the delivery of such a wide range of
476
This work was supported by grants
from the National Institutes of Health
(GM42336 and GM33301 to W. E. B.,
GM49497 to I. A. W.), the TobaccoRelated Disease Research Program of
California (TRP0222) to W. E. B. and the
Lucille B. Markey Charitable Trust.
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