From the
Department of Biochemistry, University of
Utah School of Medicine, Salt Lake City, Utah 84132 and the
Biology Department, 463 Brookhaven National
Laboratory, Upton, New York 11973-5000
Received for publication, March 13, 2003 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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The recognition of ubiquitylated proteins is frequently mediated by conserved ubiquitin binding modules, which include the UIM1 (ubiquitin interacting motif) (4, 5), UBA (ubiquitin-associated domain) (69), UEV (ubiquitin E2 variant domain) (1013), NZF (npl4 zinc finger domain) (14), and CUE (coupling of ubiquitin conjugation to ER degradation domain) (15, 16). Each of these motifs can bind ubiquitin in vitro, and the motifs are used in a modular fashion to add ubiquitin binding activities to a large variety of multifunctional proteins. Thus, understanding how ubiquitylated proteins are recognized and sorted within cells will require a full description of the structures and ubiquitin interactions of the different conserved ubiquitin binding modules.
The UIM was originally identified based upon studies of the S5a subunit of
the 19 S regulator in the human 26 S proteasome
(17). Biochemical and
mutational analyses revealed that S5a (also called rpn10) contains two copies
of a 30-residue sequence motif (initially denoted pUbS) that can bind
ubiquitylated protein and polyubiquitin chains
(5,
17). The same region of S5a
also appears to mediate contacts with ubiquitin-like proteins
(18,
19). The pUbS motifs have
hydrophobic core sequences composed of alternating large and small residues
(Leu-Ala-Leu-Ala-Leu) that are flanked on both sides by patches of acidic
residues. Sequence analyses and iterative data base searches based upon the
original pUbS motif have been used to define a more general UIM, which is
found in a number of different proteins that function in a variety of
biological pathways (4). These
sequence analyses have also provided a more precise definition of the UIM as a
20 residue sequence corresponding to the consensus:
X-Ac-Ac-Ac-Ac-
-X-X-Ala-X-X-X-Ser-X-X-Ac-X-X-X-X,
where
represents a large hydrophobic residue (typically Leu), Ac
represents an acidic residue (Glu, Asp), and X represents residues
that are less well conserved
(4).
UIMs are particularly prevalent in proteins that function in the pathways of endocytosis and vacuolar protein sorting (4). These two linked pathways serve to sort membrane-associated proteins and their cargo from the plasma membrane (or Golgi) for eventual destruction (or localization) in the lysosome (yeast vacuole). Unlike proteasomal protein targeting, which requires at least a tetraubiquitin chain (20), monoubiquitylation is sufficient to mark proteins for both endocytosis and lysosomal trafficking (2128).
Endocytic proteins that contain UIMs include the epsins (yeast Ent1p, Ent2p), Eps15, and Eps15R (yeast Ede1p) (4). These proteins are all required for endocytosis of receptor: ligand complexes, including the complex of the epidermal growth factor (EGF) with its receptor (EGFR) (27, 2932). Recent work from several laboratories has demonstrated that the UIMs in these proteins can bind ubiquitin in vitro and play essential roles in vivo, as deletion or mutation of the UIM sequences blocks receptor internalization (26, 27). The EGFR is ubiquitylated upon stimulation, and the Eps15 and Eps15R proteins are also phosphorylated and monoubiquitylated (26, 33, 34). Strikingly, Polo et al. found that mutating either of the two UIMs found in Eps15 prevented Eps15 monoubiquitylation (26). Thus, it appears that UIMs can both bind ubiquitin and also direct protein ubiquitylation, although the relationship between these two activities is not yet fully understood.
Upon internalization, ubiquitylated receptors can be sorted through the endosomal system to the lysosome via the vacuolar protein sorting pathway (reviewed in Ref. 35). UIM-containing proteins required for vacuolar protein sorting include Hrs (yeast Vps27p), Stam1 (yeast Hse1), and Stam2 (4). There appear to be strong parallels between the requirements for endocytosis and vacuolar protein sorting because the protein substrates of both pathways are ubiquitylated, the UIM domains of proteins in the pathway are required for proper sorting of substrates, and because Hrs, like Eps15, also becomes ubiquitylated in a process that again depends upon the integrity of its own UIM (2628,36). Interestingly, vesicle formation during cellular vacuolar protein sorting also appears to be intimately related to another ubiquitin-dependent process; the budding of many enveloped viruses (reviewed in Ref. 37). For example, we have recently shown that Hrs protein fragments, when fused to the C-terminal end of the structural HIV-1 Gag protein, can rescue the budding of virus-like particles that lack their cis-acting signals normally required for efficient virus budding.2
The prevalence of the UIM and its important role in the monoubiquitin-dependent processes of endocytosis and vacuolar protein sorting has led us, and others (19, 39), to study the detailed biochemical and structural basis for ubiquitin-UIM interactions. Toward this end, we have characterized the ubiquitin binding properties of UIM peptides from Eps15, Vps27p, Hrs, Stam-1, and Stam-2, and determined the high resolution crystal structure of the second UIM from Vps27p.
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EXPERIMENTAL PROCEDURES |
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GST-UIM fusion peptides were expressed in DH5 Escherichia
coli cells. Protein expression was induced with 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside (OD600 =
0.4), and after 4 h at 23 °C the cells were harvested by centrifugation (4
°C), resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 150
mM NaCl, 5 mM
-mercaptoethanol, supplemented with
Complete Protease Inhibitor tablets (Roche Applied Science)), lysed with
lysozyme (20 µg/ml) and sonication, and the supernatants were clarified by
centrifugation at 40,000 x g for 30 min. The resulting soluble
extracts were held on ice and used immediately to minimize protein
degradation.
UIM peptides used in NMR chemical shift perturbation and crystallization experiments were made by FMOC (N-(9-fluorenyl)methoxycarbonyl) solid-phase synthesis, purified using reverse phase high performance liquid chromatography, and confirmed by MALDI mass spectrometry.
Ubiquitin was expressed and purified as described (41, 42). 15N-labeled ubiquitin was expressed and purified using the same protocol except that it was expressed in M9 minimal media with 15NH4Cl as the sole source of nitrogen.
Biosensor Binding Experiments
Biosensor binding experiments were performed at 10 and 18 °C on a
BIACORE 3000 using a CM5 research-grade sensor chip derivatized with anti-GST
antibodies (40). GST-UIM
fusion peptides or GST alone (negative control) were captured from soluble
E. coli lysates at final densities of 1.12.3 kRU. Ubiquitin in
running buffers (see figure legends) was flowed over the GST-UIM and GST
surfaces at concentrations of 0, 4.1, 12.3, 37.0, 111, 333, and 1000
µM. Binding responses were recorded and globally fit to simple
1:1 binding models using the CLAMP software
(43).
Chemical Shift Perturbation Mapping Experiments
Chemical shift perturbation experiments were performed at 18 °Con a
Varian Inova 600 MHz spectrometer equipped with a triple-resonance
1H/13C/15N probe and z-axis pulsed
field gradient capability. Both peptide and ubiquitin samples were dissolved
in NMR buffer (90% 1H2O/10% 2H2O
containing 20 mM sodium phosphate pH 6.0, 10 mM NaCl),
and the unlabeled UIM peptides (10 mM) were titrated into 0.8
mM 15N-labeled ubiquitin at final molar ratios of 0:1,
0.25:1, 0.5:1, and 1:1 (UIM:Ubiquitin). 1H/15N HSQC
spectra (44) were collected at
the different titration points, processed using FELIX 97 (MSI), and analyzed
using SPARKY (T. D. Goddard and D. G. Kneller, University of California, San
Francisco). Normalized chemical shift changes (
) were calculated using
the equation:
= 25[(
HN)2 +
(
N/5)2]0.5
(45,
46). Amide chemical shift
assignments for human ubiquitin were obtained from the VLI Research, Inc.
(website:
www.vli-research.com/ubshifts.htm).
Vps27p Crystallization
Vps27p-2 UIM (Table II) was
crystallized under two slightly different conditions using the sitting drop
vapor diffusion method at 23 °C. Crystal form I was grown by mixing 3
µl of protein solution containing 1.6 mM Vps27p-2 and 0.8
mM selenomethionine-substituted ubiquitin (50 mM Tris,
pH 7.5, 150 mM NaCl) with 2 µl of reservoir solution (0.1
M imidazole, pH 7.6, 0.2 M zinc acetate, and 26%
1,4-butanediol). Crystal form II grew from the same protein solution as form I
when 3 µl of protein were mixed with 2 µl of a reservoir solution
comprised of 0.08 M sodium cacodylate pH 6.5, 0.16 M
zinc acetate, 10.4% polyethylene glycol-8000, and 20% glycerol. Both forms
crystallized in space group P6222 with essentially identical unit
cell dimensions a = b = 34.9 Å, c = 64.2
Å, indicating that the two crystal forms are isomorphous. Although the
initial goal was to crystallize a complex of UIM peptide with ubiquitin, both
crystal forms were found to contain only the Vps27p-2 peptide. Subsequently,
we confirmed that crystals with the same morphology could be grown in the
absence of ubiquitin under the same buffer conditions that grew crystals in
the presence of ubiquitin, and that these crystals diffract equally well and
index according to the same space group and cell dimensions. Data used in the
analyses described here were from a form I crystal grown in the presence of
ubiquitin and a form II crystal grown in the absence of ubiquitin.
Structure Determination
Crystallographic Data Collection and ProcessingData were
collected from single crystals of each crystal form. Crystals were suspended
in a nylon loop, rapidly cooled by plunging into liquid nitrogen, and
maintained at 100 K for data collection. Prior to cooling, the form I crystal
was soaked in reservoir solution made up with 15% glycerol. Form I crystal
data were collected at beamline x12c at the National Synchrotron Light Source,
Brookhaven National Lab at a wavelength of 0.979 Å whereas form II
crystal data were collected using a rotating anode x-ray source ( =
1.5418 Å). All data were integrated and scaled in the HKL package
(47). Data collection and
processing statistics are presented in
Table IV.
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Phase Determination and Structure RefinementData collected
from the form I crystal showed a large anomalous signal, and the program SOLVE
(48) was used to determine
phases and locations for three zinc atoms using the single-wavelength
anomalous diffraction (SAD) method. Phases were refined and a polyalanine
helix model was calculated in RESOLVE
(48). The resulting map showed
clear continuous density that allowed for the modeling of a 20-residue
-helix. Model building was performed in the program O
(49) and successive rounds of
structure refinement were completed in REFMAC5
(50), as provided in the CCP4
suite (51). Refinement
statistics are reported in Table
IV. Figures were created in MOLSCRIPT
(52) and PyMOL (DeLano
Scientific;
www.pymol.org).
Analytical UltracentrifugationEquilibrium ultracentrifugation experiments were performed on a Beckman Optima XL-A analytical ultracentrifuge. Protein samples of Vps27p-2 UIM (60220 µM) in 50 mM Tris pH 7.5, 150 mM NaCl were centrifuged at 20 °C at a rotor speed of 52,000 rpm. Absorbance measurements were recorded at 230 nm at 0.001-cm intervals every 4 h, until a stable protein distribution indicated that equilibrium had been achieved. Absorbance data were averaged and corrected for background absorbance against a buffer blank.
Equilibrium data from three different concentration distributions were simultaneously fit to a single homogenous monomer species model (53) using non-linear least squares techniques and the analysis program NONLIN (54). For these calculations, the partial specific volume was derived from the Vps27p-2 UIM sequence and estimated to be 0.7143 ml g1 at 20 °C (55).
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RESULTS AND DISCUSSION |
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As the measured ubiquitin binding affinities for the different minimal UIM peptides were surprisingly weak (Table II), we considered the possibility that adding flanking residues to either side of the minimal UIM sequences might be necessary to complete the motif and/or to negate "end" effects. This possibility was tested using longer constructs corresponding to predicted helices that encompassed the minimal UIM sequences from human Hrs, Stam1, and Stam2 (denoted Hrs-B, Stam1-B, and Stam2-B, respectively). These three proteins were chosen for further study because we are particularly interested in the roles of ubiquitin in vacuolar protein sorting and HIV budding. Representative ubiquitin binding data are shown in Fig. 1; and binding data for all of the longer constructs are summarized in Table II. Notably, the addition of 59 extra residues on either side of the minimal UIMs of Hrs and Stam-1 increased ubiquitin-binding affinity 2 to 6-fold (the comparison was not made for Stam-2). Moreover, all three of the longer UIM constructs bound ubiquitin with moderate (and similar) affinities (Kd = 150300 µM). We therefore conclude that under these experimental conditions, the residues that normally flank minimal UIMs can enhance ubiquitin binding either through direct interactions or indirectly by stabilizing the helical conformation.
Several other groups have recently reported that isolated UIM peptides can bind specifically to free monomeric ubiquitin (19, 26, 28, 39), and our experimental results provide further confirmation of this fact. Moreover, two groups have previously quantified the ubiquitin binding affinities of different UIM-containing Hrs constructs and all of the results are in good accord. Specifically, Shekhtman and Coburn (39) used NMR chemical shift titrations to estimate that ubiquitin bound with a dissociation constant of 230 ± 50 µM to a UIM peptide corresponding to Hrs residues 257278, and Raiborg et al. (28) performed biosensor experiments to show that ubiquitin bound to an immobilized Hrs fragment (residues 1289) with a dissociation constant of 300 µM. Our estimates of the Hrs-B:ubiquitin dissociation constant under low salt conditions agree very well with these published estimates (250290 µM depending upon the pH, see Tables II and III). Furthermore, the fact that Hrs-B, Stam1-B, and Stam2-B UIM peptides all bound ubiquitin with similar affinities despite significant sequence variation at the non-conserved UIM positions suggests that canonical UIMs can be expected to bind ubiquitin with dissociation constants in the 150300 µM range. The presence of multiple UIMs in most UIM-containing proteins (4) and the known association of some of these proteins (5658), suggests that this inherently modest affinity has the potential to be greatly enhanced through cooperative binding to multiply ubiquitylated proteins or complexes.
Role of Conserved UIM Residues in Ubiquitin BindingThe relative contributions of different UIM sequence elements to ubiquitin binding affinity were examined in mutational studies of the Hrs-B UIM peptide. Guided by the initial description of sequence conservation in the UIM (4), we selected the four most conserved sequence elements for mutagenesis and ubiquitin binding studies. These were: 1) the N-proximal acidic patch (tested by a 259EEEE262 to AAAA mutation in Hrs-B, denoted Hrs-B259EEEE/AAAA), 2) the nearly invariant alanine residue at UIM position 9 (Hrs-B266A/G), 3) the nearly invariant serine at UIM position 13 (Hrs-B270S/A), and the conserved glutamate residue at UIM position 16 (Hrs-B273E/A).
As shown in Fig. 1B
and summarized in Table III,
all of the mutations in conserved UIM elements reduced the affinity of
ubiquitin binding, although the magnitudes of the effects differed
significantly. Specifically, mutation of the conserved N-proximal acidic
element eliminated all detectable ubiquitin binding, mutation of the nearly
invariant alanine at UIM position 9 severely reduced (but did not eliminate)
ubiquitin binding (10-fold reduction), whereas the Hrs-B273E/A
and Hrs-B270S/A mutations reduced ubiquitin binding only 2.5- and
1.5-fold, respectively. The modest reduction in the magnitude of ubiquitin
binding for the Hrs-B270S/A mutation is somewhat surprising given
the very high degree of conservation of this serine (>98% in putative UIM
sequences). However, a Ser to Ala substitution is rather conservative and so
may be less disruptive than alternative substitutions at this position.
Indeed, there is only one predicted naturally occurring UIM in which a serine
is not found at position 13, and in that case there is also a Ser to Ala
substitution (4). In summary,
our experiments strongly support the idea that the different sequence elements
of the UIM are conserved, at least in part, because they contribute directly
or indirectly to the affinity of ubiquitin binding.
Although we are the first to quantify the contributions of different UIM sequence elements to ubiquitin binding, others have tested the functional importance of several conserved UIM residues (2628). Not surprisingly, these studies all support the idea that conserved UIM residues play functionally important roles. The experiments that are most directly relevant to ours demonstrated that mutation of the conserved serine residue at UIM position 13 (to either Asp or Glu) eliminated ubiquitin binding to Hrs and Vps27p UIM peptides in GST pull-down experiments in vitro, and also blocked the functional sorting of ubiquitylated transferrin receptors by Hrs (28) and of carboxypeptidase S by Vps27p (27) in vivo. We are not aware of any other functional tests of single UIM point mutations, but our binding experiments make the strong prediction that mutation of either of the two conserved acidic patches or of the conserved Ala residue at UIM position 9 should also result in loss of UIM function.
The Ubiquitin Binding Site of UIM
Peptides1H/15N NMR chemical shift
perturbation experiments were used to map the ubiquitin binding sites for the
Hrs-A, Hrs-B, Vps27p-1, Vps27p-2, Stam1-A, and Stam1-B UIM peptides
(Fig. 2 and data not shown).
Representative data showing the changes in the 1H/15N
HSQC spectra of ubiquitin upon titration of 01 equivalent Hrs-A are
provided in Fig. 2A.
14/70 observable ubiquitin backbone amide resonances shifted significantly
upon Hrs-A binding (
2), and the positions of the shifted residues
are shown mapped onto the structure of ubiquitin in
Fig. 2B.
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As summarized in Figs. 2,
BE, all six of the UIM peptides that we tested
behaved very similarly. All of the UIM peptides bound in fast exchange and
shifted a very similar set of ubiquitin resonances. The shifted residues
generally clustered about the C-terminal three strands of the ubiquitin
-sheet, indicating that this is the UIM binding surface. This surface
corresponds very closely to the I44 surface of ubiquitin
(Fig. 2F), which has
previously been shown to function in endocytosis, proteasomal degradation, and
HIV budding (59,
60). This surface of ubiquitin
includes the exposed hydrophobic side chains of Leu8,
Leu43, Ile44, Leu50, Leu69, and
Leu71, which could presumably interact with a complementary
hydrophobic surface on the UIM (discussed below). Ubiquitin also displays
three basic side chains; Arg42, Lys48, Arg72,
in this region, and we speculate that these may interact with the two
conserved acidic patches within the UIM. Interestingly, the Lys48
side chain is the site used to make polyubiquitin chains that target proteins
for proteasomal degradation. We therefore anticipate that covalent attachment
of additional ubiquitin moieties to this site could have a significant effect
on UIM binding. Indeed, there is some evidence that isolated UIMs can bind
more tightly to polyubiquitin chains than to monomeric ubiquitin molecules
(5,
17,
26,
41,
61), although it is difficult
to rule out possible avidity effects and/or the effect of multiple ubiquitin:
UIM contact sites in these studies.
Our experiments are in excellent agreement with two recently published chemical shift mapping studies of the ubiquitin binding sites of another Hrs UIM peptide (corresponding to Hrs residues 257278) (39) and of the hS5a protein (which contains two UIM sequences) (19). In both cases, the mapped UIM binding sites on ubiquitin correspond very closely to the site that we mapped for the six UIM peptides that we studied. This agreement lends further credence to the idea that all UIMs adopt very similar structures and bind ubiquitin in very similar ways.
Our UIM mapping studies are also consistent with the observation that an I44A mutation in ubiquitin abolished the binding of UIM peptides from Ent1p and Vps27p as assayed by co-affinity purification, whereas a F4A mutation in ubiquitin had no effect on UIM binding (27). As shown in Fig. 2B, the ubiquitin Ile44 residue is in the center of our mapped UIM binding sites, whereas the Phe4 residue is located on the opposite side of the molecule (not shown). The fact that multiple UIMs from proteins that function in the same biological pathway can bind on the same surface of ubiquitin suggests the possibility that they may compete for binding and that this could provide a mechanism for "passing" ubiquitylated substrates along a pathway. Furthermore, not only do UIM sequences bind to this surface of ubiquitin, but two other ubiquitin binding motifs whose ubiquitin interaction surfaces have been characterized (Tsg101 UEV, and NZF) also bind to this same surface (13).3 Thus, it seems likely that competition for binding to this functionally critical surface of ubiquitin must play an important role in the recognition and trafficking of ubiquitylated proteins.
Structure of the Vps27p-2 UIMIn an attempt to characterize the UIM in structural detail, we surveyed crystallization conditions for the various UIM peptides. The Vps27p-2 peptide was crystallized in space group P6222 (a = 34.9 Å, c = 64.2 Å) and x-ray diffraction data were collected to high resolution (1.45 Å). Phases were determined using the anomalous signals from the three zinc atoms in the crystal lattice. After solvent flattening, the electron density map showed clear, continuous density that was readily interpretable. The final refined model includes the entire Vps27p-2 peptide (Vps27 residues 301320, corresponding to UIM consensus residues 120). Statistics for data collection and structure refinement are summarized in Table IV.
Previous sequence analyses have suggested that the UIM adopts a helical
conformation that can be embedded in a variety of larger domain(s)
(4,
5). As shown in
Fig. 3A, Vps27p-2 adopts an
-helical structure along its length, except for the two N-terminal
residues, Glu301 and Glu302, which adopt extended
conformations. The helix is markedly amphipathic, with a hydrophobic stripe
along one side (Fig.
3A). The crystal structure is consistent with circular
dichroism (CD) spectroscopy measurements of Vps27p-2, which indicated a
transient or partial helical conformation. In both the presence and absence of
ubiquitin, this peptide displayed
40%
-helicity (data not shown).
This agrees well with previous CD and NMR experiments conducted on the Hrs
UIM, where it was seen that the Hrs UIM peptide has 45%
-helicity that
is concentrated in the central residues of the conserved UIM peptide
(39).
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Unexpectedly, the Vps27p-2 UIM crystallized as a left-handed, antiparallel four-helix bundle, with the hydrophobic face of each amphipathic helix packing into the middle of the bundle. Intriguingly, three of the four most highly conserved residues in the UIM (Leu306, Ala309, and Ser313 in Vps27p-2) all lie on the same face of the helix and make homotypic interactions with their symmetry related mates in the center of each helix. Specifically, the Leu306 side chains pack against related Leu306 side chains across the bundle, the Ala309 residues contact related Ala309 residues on adjacent helices, and the Ser-313 side chains form water-mediated hydrogen bonds with related Ser313 side chains on adjacent helices.
Oligomeric State of Vps27p-2 UIM in SolutionThe tetrameric
structure of Vps27p-2 in the crystal raised the possibility that the Vps27p-2
peptide might also oligomerize in solution. Equilibrium analytical
ultracentrifugation experiments were therefore performed to determine the
oligomeric state of Vps27p-2 in solution
(Fig. 4). Centrifugation data
from three different UIM concentrations (60220 µM) were
satisfactorily fit to a single species model, and the estimated molecular
weight for the single species was 2279 ± 100 Da, which agrees very well
with the calculated molecular weight of the Vps27p-2 monomer (2243 Da). These
data demonstrate that Vps27p-2 UIM does not form higher order species under
the solution conditions tested. As this experiment sampled Vps27p-2
concentrations as high as 1 mM, this implies that the isolated
Vps27p-2 tetramer, if it can exist in solution at all, cannot be a very stable
structure.
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Potential Biological Implications of the Vps27p Tetramer The most important question raised by our work is whether or not the Vps27p-2 UIM tetramer seen in the crystal is a biologically relevant assembly. It is not currently possible to answer this question definitively, and reasonable arguments can be made to support both sides of the question. On the negative side, the tetrameric packing of the Vps27p-2 UIM may simply reflect the fact that an amphipathic helix is being forced out of solution by the conditions of crystallization, and that the hydrophobic face of this helix, which might normally be used to bind ubiquitin, will pack preferentially against itself (as occurs in the tetramer). Moreover, the fact that the Vps27p-2 peptide does not stably associate in solution supports the idea that the tetrameric Vps27p-2 structure is not an energetically favorable one, at least under the conditions tested thus far. Importantly, when the biosensor and analytical ultracentrifugation binding experiments were repeated in the presence of zinc chloride at concentrations of 10 µM and 200 mM, no change was observed in the oligomeric state of Vps27p-2, and only small changes recorded in the binding affinity for ubiquitin (Fig. 1C and data not shown). The results of these experiments show that zinc binding does not provide important contributions to the affinity of ubiquitin for Vps27p-2 or to the oligomeric state of Vps27p-2. This is consistent with the structure, since the zinc ions mediate contacts between adjacent tetramers in the crystal lattice, and do not appear well positioned to directly stabilize the tetrameric association.
In support of the possible biological relevance of UIM tetramerization, it is striking that many of the intramolecular packing interactions within the Vps27p-2 tetramer are formed by highly conserved UIM residues. Moreover, oligomerization might explain why most UIM-containing proteins have multiple copies of the motif (4). Proteins that contain multiple canonical UIM sequences include epsin (three UIMs), epsin2 (two), Eps15 (two), Eps15R (two), Vps27p (two), hS5a (two), Ent1p (two), Ent2p (two), Vps27p (two) (4). Intramolecular UIM association should be more favorable than intermolecular tetramerization, and the tandem UIMs found in many proteins could, in principle, pack in the antiparallel orientation seen in the crystal. In addition, many UIM-containing proteins are known to associate with other proteins that also have UIMs. For example, Hrs associates with the Stam proteins (56) and with Eps15 (58), and Eps15 also associates with epsins (32, 63). Thus, there is the potential for "mixing and matching" of strands in the tetrameric structures, as is seen in other biological coiled-coil systems such as the SNARE complexes (64) and in the networks formed by various transcription factors (65, 66).
If UIM tetramerization is indeed biologically relevant, the next important question is whether tetramerization promotes or represses ubiquitin binding. Although neither possibility can be ruled out at this stage, our preferred possibility is that UIM tetramerization might serve to prevent ubiquitin binding, and thereby allow UIM systems to be regulatable. In this model, conserved UIM residues would perform important functions in both the repressed state (in stabilizing the UIM tetramer) and activated state (in binding ubiquitin). We note that Eps15 is phosphorylated by the ligand-activated EGFR at a site just two residues upstream of its first UIM (Tyr850) (67). This phosphorylation is required for ligand-regulated endocytosis, and we speculate that it might destabilize a tetramer, thereby allowing the first Eps15 UIM to bind ubiquitin.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health
(to W. I. S. and C. P. H.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence may be addressed: Dept. of Biochemistry, University of Utah, Salt Lake City, UT 84132. Tel.: 801-585-5402; Fax: 801-581-7959; E-mail: wes{at}biochem.utah.edu.
|| To whom correspondence may be addressed: Dept. of Biochemistry, University of Utah, Salt Lake City, UT 84132. Tel.: 801-585-5536; Fax: 801-581-7959; E-mail: chris{at}biochem.utah.edu.
1 The abbreviations used are: UIM, ubiquitin-interacting motif; EGF,
epidermal growth factor; GST, glutathione S-transferase; MALDI,
matrix-assisted laser desorption/ionization; RMSD, root mean-squared
deviation; Vps, vacuolar protein sorting.
2 Pornillos, O., Higginson, D. S., Stray, K. M., Fisher, R. D., Garrus, J.
E., Payne, M., He, O.-P., Wang, H. E., Morham, S. G., and Sundquist, W. I.
(2003) J. Cell Biol., in press.
3 Wang, B., Alam, S. L., Meyer, H. H., Payne, M., Stemmler, T. L., Davis, D.
R., and Sundquist, W. I. (2003) J. Biol. Chem. 278,
2022520234.
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ACKNOWLEDGMENTS |
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REFERENCES |
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