Correspondence to: Margaret S. Robinson, University of Cambridge, CIMR, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK. Tel:44 1223 330163 Fax:44 1223 762640 E-mail:msr12{at}mole.bio.cam.ac.uk.
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Abstract |
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We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH2-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal "ear" domain of -adaptin. However, unlike
-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the
-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.
Key Words: GGA, AP-1, vesicle coat, membrane traffic, protein sorting
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Introduction |
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The major components of clathrin-coated vesicles are clathrin and adaptor or AP complexes. The clathrin provides the scaffold that deforms the membrane into a vesicle, while the adaptor complexes select the vesicle cargo and also recruit accessory proteins to the site of vesicle formation. There are two adaptor complexes associated with clathrin: AP-1, which is found at the TGN, and AP-2, which is found at the plasma membrane. More recently, two additional AP complexes have been described, AP-3 (, ß1, µ1, and
1; AP-2 contains
, ß2, µ2, and
2; AP-3 contains
, ß3, µ3, and
3; and AP-4 contains
, ß4, µ4, and
4. The four subunits in the four complexes show homology to their counterparts in the other three complexes, but in the case of the
,
,
, and
subunits, the homology is restricted to the first 600 amino acids. This conserved NH2-terminal domain is followed by a hinge domain of 100200 amino acids, and then by a completely divergent COOH-terminal appendage or "ear" domain of 100300 amino acids (
-adaptin or to interact with one of the ear domain binding partners (
Although less is known about the requirements for clathrin-coated vesicle formation at the TGN, it is likely that accessory proteins are also recruited onto the membrane by AP-1. To date, only one such protein has been identified, -synergin, which binds directly to the ear domain of
-adaptin (
-synergin is an EH1 (for Eps15 homology) domain-containing protein, and by analogy to Eps15 and other EH domain-containing proteins, the EH domain of
-synergin probably binds to an as yet unidentified additional accessory protein(s) containing the tripeptide NPF (
While we were searching through the EST database for homologues of adaptor subunits, we found several sequences that showed significant homology to the ear domain of -adaptin. This homology was restricted to the last 120130 amino acids, which includes the
-synergin binding site. The NH2-terminal regions of the novel sequences contain a motif called a VHS domain (
We have cloned and characterized three novel mammalian proteins that contain both a -adaptin ear homology domain and a VHS domain. While this manuscript was in preparation, we found out that
ear-containing, ARF-binding proteins. In addition to the three mammalian GGAs, we have also identified two members of this family in the budding yeast S. cerevisiae. Like the mammalian GGAs, the yeast proteins also contain a
-adaptin ear homology domain and a VHS domain. Deletion of the two GGA genes in yeast has provided further insights into the function of the GGA protein family.
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Materials and Methods |
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Cloning and Sequencing
Several sequences within the EST database were identified that had significant homology to the COOH-terminal ear domain of -adaptin. Clones were obtained from the IMAGE Consortium, sequenced, and expressed as recombinant fusion proteins for the production of antibodies. Most molecular biology techniques were carried out as described by
Antibody Production
To construct glutathione S-transferase (GST) fusion proteins of GGA1 and GGA2, amino acids 289508 and 567639 of GGA1, and 305482 of GGA2, were amplified by PCR and ligated into pGEX-4T-1 (Pharmacia Biotech), and expression of the fusion protein was induced in MC1061 cells. The GGA1 fusion proteins were both partially soluble and were purified using glutathione-Sepharose affinity chromatography (Pharmacia Biotech). The GGA2 fusion protein was found to be insoluble and was purified from inclusion body preparations as previously described (-adaptin (described by
Expression of Epitope-tagged GGA2
An epitope-tagged version of full-length GGA2 was constructed by the insertion of the 8 amino acid (DYKDDDDK) FLAG tag at the COOH terminus and ligation into the vector pSTAR, which contains a tetracycline-inducible promoter. Transfection of normal rat kidney (NRK) fibroblasts was performed using Fugene reagent (Life Technologies Inc.), and stably transfected cells were selected with G418 (Boehringer Mannheim Corp.). Expression of the epitope-tagged GGA2 was induced by the overnight addition of 10 mM deoxycycline to the culture medium. By immunofluorescence, >90% of transfected NRK cells stained positive with M5, an mAb against the FLAG epitope (Sigma Chemical Co.).
Immunoprecipitations and Western Blotting
Immunoprecipitations were carried out on HeLa cell extracts under nondenaturing conditions, as previously described (
Immunofluorescence and Immunoelectron Microscopy
NRK cells, either nontransfected or stably expressing FLAG-tagged human GGA2, were fixed with 3% paraformaldehyde, followed by 0.1% saponin as previously described (-adaptin (
-synergin (
For immunoelectron microscopy, NRK cells were fixed either intact or after permeabilization by immersion in liquid N2 and incubation with pig brain cytosol plus ATP, an ATP regenerating system, and GTPS, as previously described (
GST Pulldown Experiments
For GST pulldown experiments, three GST fusion proteins were constructed as described above. GST-GGA1 contains amino acids 468639 of human GGA1, GST-GGA2 contains amino acids 454613 of human GGA2, and GST- contains amino acids 706823 of mouse
-adaptin (
Yeast Knockout and Rescue Experiments
The GGA-deficient strains were constructed in the YPH500 strain (
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For rescue experiments, wild-type GGA1 and GGA2 were cloned by PCR from genomic DNA prepared from YPH500. BamHI and PstI restriction sites in the primers allowed for rapid cloning into the CEN vectors pRS414 (for GGA1) and pRS415 (for GGA2), allowing the two genes to be expressed under their own promoters at approximately endogenous levels. These constructs were transformed into the JYY3 gga1/gga2
strain, as well as control strains, and the transformants selected on -trp or -leu plates. Rescue of the JHY3 strain by a mammalian GGA was also tested, using mammalian GGA2 expressed via the VPS5 promoter. Primers were designed to amplify the full open reading frame of human GGA2, incorporating the restriction sites NcoI and XhoI. The resulting PCR product was cloned into VPS5-424, replacing the VPS5 coding region with the mammalian cDNA. This construct was transformed into the gga1
/gga2
strain, as well as control strains, and the transformants were selected on -trp plates.
The single and double knockout strains were first tested for their ability to sort and process carboxypeptidase Y (CPY), using the CPY sorting assay described by
Online Supplemental Information
The online version of this article includes text and figures that accompany the information presented here and is available at http://www.jcb.org/cgi/content/full/149/1/67/DC1.
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Results |
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Identification of a Novel Family of Proteins
In a search for novel proteins related to AP components, sequences were identified within the nonredundant and EST databases that showed significant homology to the COOH-terminal ear domain of -adaptin, although their NH2-terminal sequences showed no homology to any of the AP subunits. However, the NH2 termini of these proteins showed homology to several other proteins, all of which contain a recently described motif known as a VHS domain (
-adaptinrelated proteins also contain VHS domains. There are at least three such proteins in humans, encoded by different genes: GGA1, GGA2, and GGA3. A full-length sequence of GGA3 is present in the nonredundant database (GenBank/EMBL/DDBJ accession number
D63876;
-adaptin isoforms,
1 and
2, and the putative
-adaptin in S. cerevisiae, Apl4p. Similar alignments were obtained with GGA2 and GGA3 (data not shown). GGA1 can be seen to have a typical VHS domain, and the homology with the
-adaptin COOH terminus extends over the entire ear domain.
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In addition to the VHS domain at the NH2 terminus and the -adaptin ear homology domain at the COOH terminus, all three proteins contain either one or two predicted coiled coil domains downstream from the VHS domain, followed by a variable domain, where the proteins show little or no homology with each other. However, the amino acid content of the variable domains is similar to that of the adaptin hinge domains, containing a high proportion of hydrophilic amino acids, prolines, and alanines. This suggests that the variable domain may function as a flexible stalk or hinge, connecting the conserved NH2-terminal and COOH-terminal domains to each other, in the same way that the adaptin ears are connected by hinges to the core or "head" of the AP complex. The three GGAs are shown diagrammatically in Fig 1 d.
Biochemical Characterization of GGA1 and GGA2
To characterize GGA1 and GGA2 further, we raised polyclonal antibodies against fragments of the proteins expressed as GST fusion proteins. On Western blots of whole HeLa cell extracts, the antibodies against GGA1 recognized a band with an apparent molecular weight of 85 kD, whereas the GGA2-specific antibodies recognized a band with an apparent molecular weight of 67 kD; thus, GGA1 runs slightly more slowly than predicted from its amino acid content, while GGA2 runs in the expected position (Fig 2 a).
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Because AP-1 is associated with clathrin-coated vesicles, we next investigated whether GGA1 and GGA2 are also associated with clathrin-coated vesicles. For this experiment, equal protein loadings of clathrin-coated vesicles purified from rat liver and a crude microsomal membrane fraction from an earlier stage in the preparation were subjected to SDS-PAGE, and Western blots were probed with antibodies specific for the AP-1 subunits, -adaptin and µ1; for the AP-2 subunit µ2; for the AP-4 subunit
; and for the AP-1 accessory protein
-synergin; as well as for GGA1 and GGA2 (Fig 2 b). As expected,
was depleted from clathrin-coated vesicles, since AP-4 is not associated with clathrin (
-adaptin, µ1, µ2, and
-synergin were all enriched. However, the antisera raised against GGA1 and GGA2 were unable to detect bands in the clathrin-coated vesicle preparation, although they labeled bands of the appropriate sizes in the microsome samples. Thus, despite their homology to
-adaptin, GGA1 and GGA2 do not appear to be associated with clathrin-coated vesicles.
To determine whether GGA1 and GGA2 are associated with the AP-1 complex or with any of its subunits, native immunoprecipitation experiments were performed. Fig 2 c shows the results of one such experiment. HeLa cell extract was immunoprecipitated under nondenaturing conditions with anti-, anti-GGA1, and anti-GGA2, as well as with anti-
as a control. The immunoprecipitates were subjected to SDS-PAGE, blotted onto nitrocellulose, and probed with antibodies specific for
,
, ß1, µ1,
1,
-synergin, GGA1, and GGA2. The results show that, as expected, anti-
brings down all the subunits of the AP-1 complex, as well as
-synergin. However,
does not coprecipitate with either GGA1 or GGA2. Conversely, antibodies against GGA1 and GGA2 do not bring down the other GGA,
-synergin, or any of the subunits of the AP-1 adaptor complex. These results indicate that GGA1 and GGA2 do not associate with each other or with the AP-1 complex.
To investigate the possibility that GGA1 and GGA2 form a complex with as yet unidentified proteins, pig brain cytosol was fractionated by gel filtration on a Superose 6 column. The column fractions were subjected to SDS-PAGE and Western blots were probed with anti-µ1, anti-GGA1, and anti-GGA2 (Fig 2 d). µ1 was detected in fractions 5163, peaking at fraction 57, which corresponds to >200 kD, consistent with its native molecular weight of 263 kD as a component of the AP-1 complex. In contrast, GGA1 and GGA2 were detected in fractions 6367, with a peak corresponding to an apparent size of ~75 kD. These results further support the claim that the GGAs do not associate with AP-1, and also indicate that the GGAs are not part of a large protein complex.
Localization of GGA1 and GGA2
The antibodies raised against GGA1 and GGA2 were used for immunofluorescence to determine the distribution of the protein in intact cells. The GGA1-specific antibodies were found to label a discrete pattern of dots in the perinuclear region of the cell (Fig 3 a). This distribution is similar to that of -adaptin or
-synergin; however, both
-adaptin and
-synergin are found not only on the TGN, but also on more peripheral membranes that have been shown to correspond to early and/or recycling endosomes (
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The AP-1 complex, like a number of other coat proteins, requires the small GTPase ARF to associate with membranes (
Although GGA1 and GGA2 have similar immunofluorescence patterns and are both BFA-sensitive, they do not coimmunoprecipitate, indicating that they are not associated with each other. To determine whether the two proteins colocalize, NRK cells stably transfected with FLAG-tagged GGA2 were double-labeled with anti-FLAG and anti-GGA1. Fig 3e and Fig f, shows that the double-labeling patterns are largely coincident, indicating that GGA1 and GGA2 are associated with the same membranes and that they may function together in the same pathway(s).
Cells were also double-labeled with antibodies against FLAG-tagged GGA2 or endogenous GGA1, together with antibodies against -adaptin,
-synergin, and TGN38. Fig 4 shows that GGA1 and GGA2 have very similar distributions to all three of these proteins, all of which are localized at least partially to the TGN. However, there are subtle differences in the labeling patterns. Thus, FLAG-tagged GGA2 (Fig 4 a) is more strictly perinuclear than
-adaptin (Fig 4 b), and the actual pattern of dots is distinct from that of
-adaptin, although the two are in close proximity (Fig 4, a and b). Similar results were obtained when COS cells were double-labeled with anti-GGA1 and the
-adaptin mAb 100/3 (data not shown). In contrast, double-labeling for GGA1 and GGA2 gave much more coincident patterns (see Fig 3e and Fig f). Fig 4c and Fig d, shows cells double-labeled for FLAG-tagged GGA2 (c) and
-synergin (d). Again,
-synergin is more dispersed than GGA2 and the two patterns of dots are somewhat different. By comparison, when cells are double-labeled for
-adaptin and
-synergin, the two patterns are virtually identical (
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GGA1 was also localized at the electron microscope level, both in intact cells and in cells that had been permeabilized by freezing and thawing, then incubated with exogenous cytosol plus ATP and GTPS. Previously, we have shown that this treatment stabilizes the membrane association of proteins that are recruited onto membranes in an ARF-dependent manner, such as AP-1 and AP-3 (
S can cause mistargeting of AP-2 (
S-treated cells was virtually identical to that in control cells, both by immunofluorescence and by immunogold EM (data not shown). The advantage of this treatment is that cell membranes are much easier to visualize in the electron microscope. Fig 5 a shows an example of such a cell labeled with anti-GGA1 followed by protein A coupled to 15-nm gold. The labeling is associated with Golgi membranes and with tubulovesicular membranes near the Golgi stack. Clathrin-coated budding profiles can often be seen in the same vicinity (Fig 5 a, arrowheads). These observations indicate that GGA1 is associated with Golgi cisternae, particularly on the trans side, and with the TGN.
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Binding Partners for -Adaptin, GGA1, and GGA2
What might be the functional significance of the homology between the COOH-terminal domains of -adaptin and the GGA family? One possibility is that they might share some of the same binding partners. To investigate this possibility, fusion proteins were constructed between GST and the COOH-terminal domains of
-adaptin, GGA1, and GGA2. GST pulldown experiments were then performed with all three constructs, as well as with GST alone as a control, using pig brain cytosol as a source of potential binding partners. Fig 6 shows that all three constructs bring down bands that can be stained with Coomassie blue. Three bands can be seen in the pulldowns using GGA1 and GGA2 fusion proteins, with apparent molecular weights of ~200, 160, and 56 kD (Fig 6, arrows). Bands of a similar size (labeled 2, 3, and 7), as well as a number of additional bands, can be seen in the pulldown using the
-adaptin fusion protein.
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To try to identify some of these proteins, the bands indicated with numbers were excised and subjected to MALDI mass spectrometry. Only two of the bands could be identified definitively: band 1 (MAP1A) and band 4 (rabaptin-5). Both of these proteins bound preferentially to the ear construct. Similarly,
-synergin, although it could not be identified as a Coomassie blue-stained band, was detectable by Western blotting and was found to bind preferentially to the
ear (data not shown). Together, these observations indicate that the
ear has multiple binding partners, some of which are shared by the GGA ears and some of which are not. Further details about the ear binding partners are available at http://www.jcb.org/ cgi/content/full/149/1/67/DC1 as supplemental information.
Yeast Homologues of the GGAs
A search of the S. cerevisiae genome revealed that there are two open reading frames that show significant homology to the mammalian GGAs, Ydr358w and Yhr108w. Fig 7 a shows the alignment of the two yeast sequences with GGA1. The two yeast proteins are 49% identical to each other, and each is ~20% identical to each of the mammalian GGAs. Like their mammalian counterparts, the yeast proteins consist of conserved NH2-terminal and COOH-terminal domains, separated by a variable hinge-like domain. In addition, both yeast open reading frames contain VHS domains and two potential coiled coil domains, as well as a -adaptin homology domain; thus, the two open reading frames are likely to encode the yeast orthologues of the mammalian proteins, and we propose that the two genes be called GGA1 (Ydr358w) and GGA2 (Yhr108w). Schematic diagrams of the two yeast proteins are shown in Fig 7 b.
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In an attempt to determine the functions of the yeast proteins, the GGA1 and GGA2 genes were deleted both singly and together. The single knockouts of either yeast GGA gene were completely viable and showed no obvious phenotype. However, the double knockout strain exhibited a mild, but significant, defect in the processing of the vacuolar hydrolase, CPY. Pro-CPY is normally synthesized as a p1 precursor in the ER, undergoes processing to p2 pro-CPY in the Golgi complex, and is then transported from a late Golgi compartment to a prevacuolar compartment by its receptor, Vps10p. Here, the pro-CPY dissociates from Vps10p and is delivered to the vacuole where it is proteolytically processed to the mature (m) form. Fig 8 a shows the result of a pulse-chase experiment to look at the processing of CPY in five different yeast strains. After a 10-min pulse with 35S-methionine and a 30-min chase, in the wild-type cells, 89% of the CPY was in the mature form, while the remaining 11% was in the p2 precursor form. Similar results were obtained in the cells where only one of the GGA genes had been deleted (gga2 or gga1
). In cells where the gene encoding the protein Vps35p had been deleted (vps35
), which results in strong CPY missorting, 86% of the CPY was in the p2 precursor form and only 14% in the mature form. In the yeast cells with both GGA genes deleted (gga1
/gga2
), 28% of the CPY was in the p2 form and 39% in the mature form, and in addition there was a pseudomature form, running between p2 and mature CPY.
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To confirm that the CPY sorting defect is a result of the double-deletion, the gga1/gga2
strain was retransformed with either wild-type GGA1 or wild-type GGA2 expressed at endogenous levels. Fig 8 b shows that GGA1 restores CPY processing back to wild-type levels, and similar results were obtained with GGA2 (data not shown). We also attempted to see whether we could recover a wild-type phenotype by transforming the cells with mammalian GGA2; however, the mammalian protein was unable to substitute for its yeast homologue (Fig 8 b).
To examine the fate of the different forms of CPY in the gga1/gga2
strain, the cells were spheroplasted to release proteins trapped inside the cell wall. Fig 8 c shows that in the wild-type cells, 98% of the CPY was retained intracellularly in the mature form. In contrast, in the vps35
cells, 86% of the CPY was secreted in the p2 form. In the gga1
/gga2
cells, 44% of the CPY was retained intracellularly, mainly in the mature form, while the rest was secreted in both the p2 and pseudomature forms. This result indicates that the gga1
/gga2
cells exhibit true missorting rather than a delay in processing and trafficking to the vacuole. Further evidence for missorting rather than delayed processing was obtained by carrying out a time course of pulse-chase experiments. Fig 8 d shows that immediately after the pulse, the CPY was in the p1 form in all three strains. After 15 min, the p1 form had disappeared in all three strains and had been replaced either by the p2 form alone in the vps35
strain, by the p2 form together with the mature form in the wild-type strain, or by the p2, pseudomature, and mature forms in the gga1
/gga2
strain. After longer chase times, the protein remained in the p2 form in the vps35
strain, whereas in both the wild-type strain and gga1
/gga2
processing was essentially complete by 30 min, although in the gga1
/gga2
strain much of the protein remained in the p2 or pseudomature form.
To characterize the nature of the defect further, we looked at several criteria that have been used to group the vacuolar protein sorting mutants into different classes. The defect in the gga1/gga2
strain appears to be specific for the classical vacuolar protein sorting pathway, since alkaline phosphatase, which uses a different, AP-3 mediated pathway to get to the vacuole (
/gga2
strain, have been shown to accumulate Vps10p in a prevacuoloar compartment, called the Class E compartment, and in these cells the Vps10p becomes proteolytically clipped and is degraded more quickly than in wild-type cells (
/gga2
cells (Fig 9, a and b), and the turnover time and electrophoretic mobility of Vps10p were also found to be normal (data not shown). Some of the vps mutants have been shown to be deficient in endocytosis, which can be observed by monitoring the uptake of the lipid-soluble styryl dye, FM4-64 (
/gga2
cells. However, the appearance of the vacuoles is different in the wild-type and gga1
/gga2
cells. We scored 43% of the gga1
/gga2
cells as having fragmented vacuoles, while 31% had one large vacuole surrounded by a number of smaller vacuoles. In contrast, when we examined the wild-type strain, >99% had 13 vacuoles of normal appearance. Thus, in addition to their CPY sorting defect, the gga1
/gga2
cells also have a vacuolar morphology defect, similar to that reported for class B and class F vps mutants (
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Discussion |
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Here, we describe a novel family of proteins, conserved between yeast and mammals, that contain an NH2-terminal VHS domain, one or two potential coiled coil domains, a variable hinge-like domain, and a COOH-terminal domain homologous to the COOH-terminal domain of -adaptin. There are at least three such proteins in mammals, GGA1, GGA2, and GGA3, and there are two in yeast, Gga1p and Gga2p.
Because of the homology between -adaptin and the GGAs, we set out first to determine whether the GGAs were associated with clathrin-coated vesicles or with the AP-1 complex. We found that neither GGA1 nor GGA2 was enriched in clathrin-coated vesicles, nor did they coimmunoprecipitate with any of the subunits of the AP-1 complex. Similarly, by both immunofluorescence and immunogold EM, the GGAs were found to have a distinct distribution from AP-1, although they were in close proximity on membranes of the TGN. In addition, deleting the two GGA genes in yeast gives a different phenotype from deleting AP-1 subunits or clathrin. Thus, yeast cells that are deficient in both Gga1p and Gga2p missort CPY, but endocytosis appears normal. In contrast, cells that are deficient in clathrin have reduced endocytosis, but sort CPY normally (
The sequences of the GGAs provide additional clues about their function. At the extreme NH2-terminal end, the proteins contain a VHS domain. This is the same position where VHS domains are found in all other VHS-containing proteins so far identified. Although the function of the VHS domain is still unknown, it seems likely, based on what is known about other domains with a similar degree of conservation, that it interacts either with other proteins or with lipids. It has been proposed that the VHS domain may participate in the association of such proteins with membranes, since a construct consisting of the first 205 amino acids of EAST, including the VHS domain (amino acids 1139), but not the coiled coil or SH3 domains, is sufficient for localization to the plasma membrane (
Downstream from the VHS domain, the GGAs all contain one or two predicted coiled coil domains. Although coiled coil domains are known to participate in proteinprotein interactions, the native molecular weight of GGA1 and GGA2, as determined by both gel filtration (Fig 2 d) and ultracentrifugation (
The third recognizable domain on the GGAs is the -adaptin ear homology domain, found at the COOH-terminal end. The
-adaptin ear has been shown to be the binding site for a number of accessory proteins that participate in the endocytic pathway (
ear plays a similar role in the AP-1 pathway. Recently, we have identified a novel protein,
-synergin, which binds to the
-adaptin ear, and here we show that the
ear binds to a number of other proteins in GST pulldown experiments. A subset of these proteins also bind to the GGA ears in GST pulldown experiments, and we are currently attempting to identify the common binding partners. At present, our working hypothesis is that the COOH-terminal domains of the GGAs, like the COOH-terminal domains of both
-adaptin and
-adaptin, serve to recruit accessory proteins onto a particular compartment, in this case the late Golgi complex and TGN.
Are the GGAs coat proteins? Although this question has yet to be formally addressed, several lines of evidence suggest that they may be. They have a punctate distribution by immunofluorescence, and in the electron microscope GGA1 is frequently seen associated with vesicular profiles. The GGAs are sensitive to BFA, and so far, most of the BFA-sensitive peripheral membrane proteins that have been identified are either ARFs, coat components (e.g., AP-1, AP-3, AP-4, and coatomer), or proteins associated with coat components (e.g., -synergin). In addition, the presence of the
-adaptin ear homology domain suggests that, like the
-adaptin ear, this domain may recruit proteins onto the membrane that are required for vesicle budding. Finally,
What might be the fate of such vesicles? The yeast knockout experiments show that deleting the two GGA genes causes cells to missort the vacuolar hydrolase CPY, providing strong evidence for a role for the GGAs in the delivery of proteins to the yeast vacuole and its mammalian equivalent, the lysosome. Over 50 vacuolar protein sorting genes in yeast have now been identified and characterized by screening for CPY missorting, and the reason that the GGA genes have not been identified as VPS genes until now is presumably because they are functionally redundant, so that both of them need to be disrupted to get a vps phenotype. The various VPS genes have been grouped into different classes depending on a number of criteria, including the strength of CPY missorting, the morphology of the vacuole, the ability of the cells to sort alkaline phosphatase, and whether or not endocytosis is defective (/gga2
cells. In addition, the gene encoding the VHS domain-containing protein Vps27p is a class E gene (
/gga2
cells, indicating that the GGA genes are not class E genes.
In addition to the CPY missorting phenotype, the gga1/gga2
strain has a severe vacuolar morphology defect. Many of the cells were found to have fragmented vacuoles, while other cells had one large vacuole surrounded by several smaller vacuoles. Fragmented vacuoles are found in the class B mutants, while large vacuoles surrounded by smaller ones are characteristic of the class F mutants (
/gga2
mutants. Thus, it is difficult to assign GGA1 and GGA2 to any of the classes of VPS genes. It is possible that they may be acting at a different step from any of the genes so far described. Further studies, making use of triple mutants, where not only GGA1 and GGA2 have been deleted, but also one of the well characterized VPS genes, should help to define the phenotype further. We also intend to knock out the GGA genes together with the genes encoding clathrin and/or the
-adaptin homologue Apl4p, to see if the phenotype is exacerbated. Since our hypothesis is that there are accessory proteins that are required for sorting to the vacuole, which can be recruited onto the membrane by binding either to
-adaptin or to one of the Gga proteins, then by knocking out all three we may completely block CPY sorting.
Although there is still much that we do not know about the GGA family of proteins, the ability to study these proteins in both mammals and yeast has allowed us to learn much more about their function than would have been possible with either system alone. Morphological studies are much easier to perform in mammalian cells than in yeast, because of their larger size and the better defined morphology of their organelles. By localizing GGA1 and GGA2 in mammalian cells at both the light and the electron microscope level, we have demonstrated that the proteins are recruited onto trans-Golgi cisternae and the TGN. However, simply localizing the proteins does not tell us their function. By analyzing the phenotype of GGA-deficient yeast, we have shown that they are involved in vacuolar protein sorting. However, a large number of VPS genes have been described in yeast, and in many cases it is not known at what stage the proteins act: whether they are required for vesicle budding, docking, or fusion, and whether they participate in trafficking from the late Golgi to a prevacuolar endosomal compartment, from the prevacuole to the vacuole, from the prevacuole back to the Golgi, or at some other step. By combining data from both yeast and mammalian systems, we can conclude that members of the GGA family are recruited late Golgi membranes and that from there they facilitate the trafficking of proteins that are destined for the vacuole or lysosome.
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: CPY, carboxypeptidase Y; EAST, epidermal growth factor receptor-associated protein with SH3 and TAM domains; EH, Eps15 homology; GGAs, Golgi-localized, ear-containing, ARF-binding proteins; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; MALDI, matrix assisted laser desorption ionization; NRK, normal rat kidney; STAM, signal transducing adaptor molecule; VHS domains, domains containing proteins Vps27p, Hrs, and STAM.
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Acknowledgements |
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We thank George Banting for anti-TGN38, Rainer Duden for yeast strains and advice, Abi Stewart for printing the electron micrographs, and Paul Luzio, John Kilmartin, Juan Bonifacino, and members of the Robinson lab for reading the manuscript and for helpful discussions.
This work was supported by grants from the Wellcome Trust and the Medical Research Council.
Submitted: 17 December 1999
Revised: 15 February 2000
Accepted: 22 February 2000
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