From the Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Hamburg University, Martinistrasse 52, D-20246 Hamburg, Germany
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ABSTRACT |
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GEF1 encodes the single CLC putative chloride channel in yeast. Its disruption leads to a defect in iron metabolism (Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J., and Eide, D. (1993) Mol. Gen. Genet. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification. We now show that gef1 cells indeed grow less well at less acidic pH. However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi. Thus, Gef1p may be important in determining Golgi pH. Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function. One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localization. The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2.
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INTRODUCTION |
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The CLC1 proteins form an evolutionarily old group of membrane proteins with homologues identified in bacteria, archaebacteria, yeast, plants, and animals (1, 2). Expression cloning of ClC-0, a voltage-gated chloride channel from Torpedo electric organ, yielded the first CLC cDNA (3). The completion of the Saccharomyces cerevisiae genome project shows that there is a single CLC gene in yeast.
Four members of the CLC family have been unambiguously shown to function as chloride channels. ClC-0 provides the high conductance of the non-innervated membrane of the Torpedo electric organ. In mammalian skeletal muscle, ClC-1 ensures a high resting chloride conductance. Mutations in its gene lead to myotonia because they render the muscle hyperexcitable (4). ClC-2 is an ubiquitously expressed chloride channel which is activated by cell swelling, hyperpolarization, or acidic pH (5, 6). The situation is less clear for ClC-5, a channel mutated in certain kidney stone diseases (7). It yields currents only in an unphysiological voltage range (8), which also applies to ClC-4.2 To explain the proteinuria observed with ClC-5 mutations, it was proposed that ClC-5 operates in vesicles of the endocytotic pathway of proximal tubules (8, 9). Functional expression is controversial for ClC-3, ClC-Ka and -Kb, and no plasma membrane currents were detected when ClC-6 and ClC-7 were expressed in Xenopus oocytes (1, 10). One possible explanation is that some CLC proteins function as chloride channels in intracellular compartments.
Many intracellular organelles such as the Golgi, lysosomes, synaptic vesicles, and endosomes contain chloride channels, which have not yet been identified at the molecular level. There is some evidence that cystic fibrosis transmembrane conductance regulator, the cystic fibrosis chloride channel, may play a role in vesicle trafficking (11). Chloride channels of intracellular organelles are thought to facilitate intravesicular acidification by providing an electric shunt for the proton ATPase. An acidic lumenal pH is not only essential for enzymatic activities in lysosomes, but also for sorting in the endocytotic pathway. The pH gradient along the exocytotic pathway may be important for the retrieval of ER proteins (12) and the sorting of secretory proteins (13, 14). Furthermore, the retrieval of certain TGN proteins from the cell surface requires endosomal acidification (15).
In yeast, a genetic screen for an iron-suppressible petite phenotype identified a CLC gene dubbed GEF1 (for glycerol/ethanol Fe-requiring, (16); to comply with the CLC nomenclature, we have also called this gene ScCLC (17)). Among four recently cloned plant CLCs from Arabidopsis, one could rescue the growth phenotype of gef1 strains on low iron medium (17). Interestingly, GEF2, whose null mutation has a similar phenotype, encodes a subunit of the vacuolar H+-ATPase (16, 18). The strong homology of the GEF1 gene to CLC chloride channel genes suggests that its gene product might provide an electrical shunt for the H+-ATPase, thereby facilitating the acidification of a common compartment. Greene et al. (16) hypothesized that this mechanism may be relevant to the vacuole which is involved in iron storage in yeast (19).
We now show that Gef1p resides in the Golgi. Cytosolic NH2 and COOH termini of the protein were subjected to alanine scanning mutagenesis. It revealed a 5-amino acid motif in the NH2 terminus that is essential for proper function. In the COOH terminus, mutating a larger domain conserved in all eukaryotic CLC homologues caused mislocalization of the mutant proteins which no longer complemented the gef1 phenotype. Growth assays on titrated media revealed an increased sensitivity of gef1 cells toward neutral pH. These results are compatible with a role for the putative chloride channel Gef1p in compartmental acidification of the Golgi apparatus.
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MATERIALS AND METHODS |
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Strains and Media--
Standard yeast media and genetic
manipulations were as described (20). LIM50 low iron selection medium
was prepared as described (21). pH-adjusted media for plates were
titrated with free Tris base resulting in a Tris concentration between
1 mM for pH 5.5 and
6 mM for pH 6.5. Bromcresol purple (30 µg/ml) was added to these plates as a pH
indicator. Methionine was generally omitted from the media to allow for
maximal expression from plasmids pDR46 and p416MET25 (see below). The
GEF1 gene disruption was introduced into the strains K700
(Mat
, HMLa, HMB
, ho, ade2-1, trp1-1, can1-100, leu2-3,
2-112, his3-11, 3-15, ura3, ssd1;(22)) and DF5a (Mat a,
lys2-801, leu2-3, 2-112, ura3-52, his3
200, trp1-1(am) (23)) by
transforming the strains with a linear DNA fragment containing 375 base
pairs of the 5' and 450 base pairs of the 3' portion of the
GEF1 open reading frame with the HIS3 marker gene inserted
between them. Gene disruption was verified by Southern blotting and
polymerase chain reaction. Western blot analysis of the Gef1p GFP
(green fluorescent protein) fusion protein and corresponding ALA
mutants was performed on strain GPY385 transformed with the
corresponding plasmids (Mat a, leu2-3, 2-112, ura3-52, trp1-289,
sst1-3, his4 or his6 pep4::LEU2 (24)).
Plasmid Construction-- All plasmids were derived from the wild-type GEF1 gene fused to the GFP cDNA (mutant S65T) via a NotI restriction site engineered in the place of the GEF1 stop codon. This construct was in pDR46, a yeast expression vector derived from pSEY8 (25). It carries the URA3 marker gene and the MET3 promoter fragment (26). Alanine scanning mutagenesis was performed by introducing windows of five alanines by polymerase chain reaction. Briefly, two fragments were amplified with primers containing the codons for the five alanines in an overlapping region and adjacent sequences that served to anchor the primer to the desired position in the GEF1 sequence. The fragments were joined by recombinant polymerase chain reaction, digested with appropriate restriction endonucleases, and ligated into the backbone. Polymerase chain reaction-derived fragments and restriction sites used for ligation were sequenced. COOH-terminal domain swapping was based on a BglII restriction site naturally occurring in rat ClC-2. A BglII site was engineered in the same region of the GEF1 gene thereby causing the mutation M652L. The mutated GEF1 gene behaved like wild-type in all assays. Since there is a methionine in position 653 the BglII site could also be used to transplant this part of the protein to the NH2 terminus by ligating it to the BamHI site of the 5' polylinker sequence of pDR46. In these constructs the same NotI site that was used for fusion of the GFP cDNA was used for linking the transplanted 3' portion and the original 5' portion of the GEF1 gene. To this end the NotI site was engineered on the original initiator ATG (actually deleting the methionine) in a way that allowed in-frame fusion of the COOH and NH2 terminus. Concatamers of different GEF1 mutants were constructed using the same NotI restriction site.
Complementation Assays-- Complementation of the gef1 phenotype, the inability to grow on iron-limited plates, was assayed in at least three independent transformations for each construct. The wild-type GEF1 GFP fusion construct was included in all experiments as a positive control. Equal aliquots from each transformation were spotted on synthetic complete selection plates lacking uracil and methionine as well as on LIM50 iron-limiting medium. Depending on the construct growth of all (wild type) or no (e.g. ALAN8, ALAC5, and ALAC7) transformants was observed on LIM50 medium.
GFP Fluorescence Assays-- The evening before fluorescence microscopy a culture was inoculated from a stationary preculture in a dilution that resulted in early logarithmic growth the next day. Because formation of fluorescent GFP is more efficient at lower temperatures (27) cultures were transferred to 4 °C for a period of 1 to 4 h. In control experiments it was ensured that this did not change the observed staining patterns. Either live cells were attached to concanavalin A-coated microscope slides (28) and viewed directly, or cells were fixed, the cell wall was digested with zymolyase (see "Immunostaining of Yeast Cells"), and spheroblasts were attached to poly-L-lysine-coated slides. The two treatments result in slightly different appearance of the GFP staining pattern but differences between constructs (e.g. wild-type and significantly changed localizations) were always much larger. GFP fluorescence was observed and photographed using a Zeiss Axiophot 1000 microscope and a FITC filter set.
FM4-64 and Quinacrine Staining Assay-- The styryl dye FM4-64 was used as described (28). Basically, we looked at steady-state labeling of the vacuole. FM4-64 fluorescence was observed using the rhodamine filter set of the Zeiss Axiophot 1000 epifluorescence microscope. The fluorescence of the dye was also visible as bright orange when using the FITC filter set allowing for a direct comparison of the FM4-64-stained and the GFP-stained structures. Quinacrine staining was performed as described (29). Cells were viewed and photographed using FITC optics.
Immunostaining of Yeast Cells--
Cells were grown to early
logarithmic phase, fixed at room temperature in freshly prepared
fixative (29) for at least 8 h, washed in 0.1 M
potassium phosphate, pH 6.5, and 1.2 M sorbitol in this
buffer (30). The cell wall was digested with Zymolyase 100T (0.3
mg/ml; ICN) and spheroblasts were washed in 1.2 M sorbitol and permeabilized by a short incubation in 1% SDS in 1.2 M
sorbitol (29). Subsequent staining with anti-FLAG M2 mouse monoclonal antibody (1 ng/µl, ABI), anti-Kar2 rabbit polyclonal antiserum (1:5,000, a gift of M. Rose), anti-c-Myc mouse monoclonal antibody 9E10
(10 ng/µl, Boehringer) and affinity-purified rabbit anti-Kex2 polyclonal antibody (1:100, a gift of Robert Fuller) followed Roberts
et al. (29). The secondary antibody was a CY3- or
FITC-conjugated anti-rabbit or anti-mouse IgG antibody raised in goat
(1:1000, Jackson). Images of immunostained cells expressing GFP fusions of Gef1p and the mutants were recorded with a Bio-Rad confocal microscope.
Western Blotting-- 15 A600 units of yeast cells were harvested by centrifugation and resuspended in 100 µl of TEA (7.5 g/liter triethanolamine, 0.38 g/liter EDTA, pH 8.9, supplemented with "Complete" protease inhibitor (Boehringer Mannheim), and 1 mM diisopropyl fluorophosphate). Cells were lysed by 5 × 30 s vortexing cycles with the help of glass beads. Extract of approximately 1 A600 unit was loaded per lane and resolved on a 10% SDS gel. Gels were blotted on nitrocellulose. Horseradish peroxidase-coupled anti-mouse secondary antibody and Amersham ECL reagents were used for detection.
Colony Immunoblotting-- Strains to be assayed for Kar2p secretion were streaked on a plate of synthetic complete medium lacking the appropriate amino acids. Streaks were overlaid with pre-wetted nitrocellulose and incubated at 30 °C overnight. The membrane was removed, washed in distilled water, and subsequently probed by standard Western blotting techniques with an anti-Kar2p rabbit polyclonal antiserum. Chemiluminescent detection used the ECL system (Amersham).
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RESULTS |
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Gef1p Is Localized to the Golgi--
To assess the subcellular
localization of Gef1p, we expressed an epitope-tagged version of the
protein in K700Pst (gef1) cells. Immunofluorescence
revealed multiple dot-like structures in the cytosol that were rather
variable in size and number (Fig. 1A). This pattern resembles
the staining pattern observed for Golgi proteins (31). A similar
pattern was observed with a construct in which the green fluorescent
protein was fused to the carboxyl terminus of Gef1p (Fig.
1B). Different expression levels (obtained by adding
different concentrations of methionine to the growth medium) gave
results varying only in signal intensity (data not shown). Thus, the
expression pattern is unlikely to be an artifact of overexpression.
Both the FLAG-tagged and the GFP-tagged constructs complemented the
growth defect of gef1 strains on iron-limiting media as
effectively as wild-type Gef1p, suggesting that they reach the
compartment in which Gef1p is normally expressed (data not shown).
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NH2- and COOH-terminal Truncations Render Gef1p
Nonfunctional--
In a search for functionally important domains in
the cytoplasmic NH2 and COOH termini of Gef1p, we truncated
the first 36 or the last 198 amino acids of the protein and made
COOH-terminal fusions to GFP (Fig.
3A, constructs b
and c). These were expressed in gef1 strain
K700Pst. Neither of the truncated proteins could complement the
growth defect of the cells on the iron-limiting LIM50 selective medium
(Fig. 3B). The GFP fluorescence pattern observed for the
truncated proteins lacked the dot-like structures obtained with WT
Gef1p (Fig. 3C). Instead, a strong perinuclear fluorescence
and staining close to the cell periphery were observed. We conclude
that both the amino and the carboxyl terminus of Gef1p are necessary
for the protein to fulfill its normal function. These regions may
contain residues important for correct localization. Alternatively, the
truncated proteins may be misfolded and retained in an early
compartment by a quality control mechanism.
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Alanine Scanning Identifies 5 Residues in the Gef1p NH2 Terminus and 25 Residues in the Gef1p COOH Terminus That Are Essential for Proper Function-- To narrow down the functionally important regions in the NH2 and COOH termini of Gef1p, we systematically moved a window of 5 alanine residues through the regions of interest. For the NH2 terminus, we scanned the whole region deleted in the experiment described above by constructing nine mutants in which 5 consecutive residues of the amino terminus were replaced by alanines (mutants ALAN1 to ALAN9, Fig. 4A). For the carboxyl terminus, we analyzed 17 such constructs (mutants ALAC1 to ALAC17).
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Co-localization of Non-functional ALA Mutants with ER-resident
Kar2p--
In yeast, retention-defective mutant forms of resident
Golgi proteins are often sorted to the vacuole (41). This is clearly not the case for our mutants. Their often perinuclear localization is
rather compatible with a localization in the ER. We therefore examined
co-localization with Kar2p, a lumenal ER chaperone of the Hsp70 family
(42). K700Pst (gef1) cells expressing either Gef1p-GFP
or two defective mutants, ALAN8 and ALAC5, were co-stained with an
anti-Kar2 antiserum (Fig. 5). The
wild-type protein was always found in structures distinct from those
stained for Kar2p (Fig. 5A), while both mutants co-localized
with this ER protein (Fig. 5, B and C). In
addition to this typical perinuclear and submembraneous fluorescence,
atypical structures were observed in cells expressing higher amounts of
mutant Gef1 proteins, especially for ALAC5. This effect was the same in
the corresponding GEF1 strain (data not shown). These
structures also stained for Kar2p and might therefore represent
abnormal ER-derived membranes. Thus, non-functional ALA mutants of
Gef1p are retained in the ER, and expression of mutant proteins leads
to abnormal Kar2p-containing structures.
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The Carboxyl-terminal Essential Domain Coincides with CBS Domain 2 and Functions in a Position-independent Manner--
Recently, Bateman
(43) and Ponting (44) identified a new class of protein domains called
CBS (for cystathione--synthase) domains. Two copies of this domain are found in the carboxyl termini of
all eukaryotic CLC homologues, and deletion of both CBS domains in
construct Gef1p
C198 resulted in a loss of function as shown above.
Interestingly, the region identified by our systematic alanine scanning
of the COOH terminus nearly exactly co-incides with the second CBS
domain (CBS2) (Fig. 4B). As expected from this scan,
deletion of CBS2 (leaving CBS1 intact) also impaired Gef1p function
(Fig. 6a). To examine whether
the first CBS domain (CBS1) is also important, we replaced residues 640 to 644 (GYVLK, located centrally in CBS1) by alanines (Fig.
6b). Again, this mutant displayed perinuclear staining and
could not restore growth under iron-limiting conditions. Thus, both CBS
domains are needed for proper function.
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The Gef1p COOH Terminus Cannot be Substituted by the ClC-2 COOH Terminus-- To elucidate the role of the Gef1p COOH terminus further, we replaced the last 128 amino acids of Gef1p by the corresponding 127 residues of the rat ClC-2 protein (Fig. 6e). Chimeric ClC-2 proteins in which the COOH terminus was replaced by that of ClC-1 still yield currents when expressed in Xenopus oocytes, although their characteristics are changed (6). In contrast, the chimeric protein Gef1p/C2 could not complement the growth defect of the gef1 strain on iron-limiting LIM50 plates, and the GFP staining pattern resembled that observed for truncated Gef1 proteins and non-functional ALA mutants. Thus, the COOH-terminal region contains essential residues that are not conserved in the corresponding sequence of ClC-2. We next fused the Gef1p CBS2 domain to the amino terminus of the chimera Gef1p/C2 (Fig. 6f). This construct complemented the growth of gef1 cells on LIM50 plates, and the associated GFP fluorescence resembled the punctate staining of WT Gef1p. ALAC mutations which destroyed proper Gef1p function when inserted into CBS2 at the COOH terminus (Fig. 4) also destroyed the function of the chimera when inserted into the CBS2 domain transplanted to the amino terminus (data not shown). This demonstrates that the effect of the transplantation is due to a specific effect of the CBS domain.
Concatamers of Wild-type and Mutant Gef1 Proteins Are Functional-- ER retention of the non-functional GEF1 mutants could result from a misfolding of the protein rather than reflect specific functions of the mutated residues. We linked mutant and wild-type proteins in a concatameric fashion in order to address this question. We reasoned that the recognition of a misfolded protein region by some quality control mechanism in the ER should not be influenced by linking it with WT Gef1p. Thus, ER retention due to misfolding should also operate on the concatamer, resulting in a non-functional protein.
However, concatamers linking mutant ALAC6 to WT Gef1p efficiently complemented the knockout phenotype irrespective of the order in which the proteins were fused (data not shown). Examining their intracellular localization by GFP fluorescence revealed that cells with almost wild-type appearance were mixed with cells that exhibited untypical staining. These findings argue against ER retention of the mutant proteins being due to misfolding.CLC Knockout Strains Display Reduced Tolerance Toward Neutral
pH--
If Gef1p is involved in pH regulation, one might expect a
growth phenotype on media adjusted to different pH values. This hypothesis was tested for two different genetic backgrounds, K700 and DF5a. Growth was assayed on synthetic complete medium titrated to
pH 5.5, 6.0, and 6.5, respectively (Fig.
7A). Two different carbon
sources, dextrose and sucrose, were used. On dextrose, both wild-type
strains were able to grow on plates titrated to all three pH values,
whereas gef1 strains could not grow on plates titrated to pH
6.5. On sucrose both wild-type strains were unable to grow on plates
titrated to pH 6.5, and the corresponding gef1 strains
stopped growing at pH 6. Growth on sucrose was probably more sensitive
toward neutral pH since invertase, the key enzyme in sucrose
metabolization, requires acidic pH for optimal function (45). These
results show that gef1 strains are less tolerant toward
neutral pH than the corresponding wild-type strains. The same result
was found when growth of the same strains was assayed on full medium
buffered with 100 mM Tris-HCl to pH 7.5. No growth of the
gef1 strains was observed whereas the corresponding
wild-type strain grew slowly.
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DISCUSSION |
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Many intracellular compartments of eukaryotic cells are acidified. An acidic intravesicular pH, and the pH gradient along endo- and exocytotic pathways, is not only important for certain enzymatic activities, but also for receptor-ligand interactions, protein sorting, and vesicle trafficking (for review, see e.g. Ref. 47). Acidification depends on a vacuolar-type H+-ATPase. This pump is electrogenic and therefore creates a transmembrane potential that in turn inhibits pumping. A parallel conductive pathway can relieve this energetic constraint on proton pumping. In higher eukaryotes, this conductance is provided by chloride channels, the molecular identity of which is still unclear.
Greene et al. (16) reported that disruption of either GEF1, a member of the CLC family of chloride channels (1, 2), or of GEF2 (VMA3), a subunit of the V-type ATPase, led to the same phenotype. This indirectly suggested that both proteins functionally interacted in the acidification of an intracellular organelle. Since the vacuolar H+-ATPase is required for the acidification of the vacuole, which is a site of iron-storage in yeast (19), it seemed reasonable to hypothesize that Gef1p resides in that organelle. However, in line with previous unpublished observations (16), our results suggest that vacuolar pH is normal in gef1 cells and that Gef1p is not (predominantly) localized to the vacuole. Moreover, iron storage in yeast was reported to not depend on vacuolar acidification (48).
On the other hand, our finding that gef1 cells are more sensitive toward neutral pH is compatible with a role of the yeast CLC in intracompartmental acidification. We have shown that Gef1p is present in the yeast Golgi apparatus, and the significant overlap with Mnt1p suggests that the medial Golgi is the predominant site of expression. It is known that the Golgi lumen is acidified, and that an increasingly acidic pH along the pathway from the ER to the TGN is important for protein localization and sorting. However, we could not detect an effect on the retention of Kar2p, an ER protein which is retained by an HDEL signal. Interestingly, Manolson et al. (49) found a novel isoform of Vph1p, the 100-kDa subunit of the V-ATPase. In contrast to Vph1p, this isoform (Stv1p) did not localize to the central vacuole. It contains two F-X-F-X-D motifs that are necessary for the efficient retention of dipeptidyl aminopeptidase A in the yeast Golgi, and may therefore be localized to that compartment (50).
The Golgi localization of Gef1p does not offer an obvious explanation for the poor growth of gef1 cells on low iron media. Radisky et al. (51) have recently characterized a gene, VPS41, that is required for vacuolar trafficking and high-affinity iron transport. In the vps41 mutant, incorporation of copper into the multi-copper oxidase Fet3p is impaired. Fet3p is an enzyme necessary for high-affinity iron uptake. Since glycosylation of Fet3p is normal in this mutant, the defect seems to lie beyond the TGN, probably in the prevacuolar endosome. Conceivably, the gef1 phenotype may also be due to a trafficking defect of some essential component of yeast iron metabolism. We could not detect an obvious effect of GEF1 disruption on the glycosylation of invertase, but this does not rule out a change in Golgi pH which may affect the sorting of some proteins. Interestingly, inhibition of the vacuolar proton pump in mammalian cells can induce retrograde transport from the TGN to the Golgi stack (52). However, the mechanism by which disruption of a putative Golgi chloride channel leads to the defect in iron metabolism of gef1 cells remains to be elucidated.
In a search for functionally important domains, we performed systematic alanine scanning experiments at the amino and carboxyl terminus. This revealed that a 5-amino acid motif in the amino terminus and a larger domain in the COOH terminus are essential for the proper function and localization of the protein. Some mutant proteins were affected in the staining pattern of the Gef1p-GFP fusion protein but remained functional. However, we cannot exclude that low level expression, which may suffice for functional complementation, still occurred at the normal site within the cell. All non-functional mutants displayed an abnormal GFP fluorescence pattern. The scan identified no mutant that seemed to be correctly localized and non-functional. However, in analogy to the many mutations analyzed in mammalian CLC channels, we expect that such phenotypes could be obtained by mutating residues in the transmembrane region.
The NH2-terminal essential motif
FVTID complies with a consensus of sorting
signals containing aromatic amino acids that are involved in localizing
proteins to the Golgi and in cycling mechanisms between the Golgi and
the plasma membrane. These include the localization determinants of the
cation-independent mannose 6-phosphate receptor in mammals
(YSKV (53)), the yeast Kex1 and Kex2
Golgi-resident proteases (YTSI and
YEFDII, respectively (41, 38)), and the internalization signal in the NH2 terminus of the mammalian
glucose transporter GLUT4 (FQQI (54)). The
consensus for these signals is -
, where
stands for an
aromatic and
for large aliphatic side chains (55). Therefore, we
additionally mutated these residues either singly or in combination
(F36A and I39G). Although expression levels had to be decreased to see
an effect of the single point mutants and only the double mutant fully
destroyed Gef1p function, these results further support the notion that
it constitutes such a motif. Whether it truly functions as a
determinant for Gef1p localization remains to be shown. It is mostly
proteins with only one transmembrane domain where similar motifs were
shown to be necessary and sufficient for localization. The issue may be
more complicated for multispanning proteins like GLUT4 where at least two localization determinants were identified (54, 56, 57). Interestingly this amino-terminal motif is highly conserved in the
mammalian ClC-3, -4, and -5 homologues (FHTID in ClC-3 and -4, FNTID
in ClC-5 as compared with FVTID in Gef1p).
For two TGN proteins, TGN38 and furin, endosomal acidification is needed for retrieval into the Golgi compartment (15). Acidification-dependent localization to the TGN depends on the cytosolic tail of these two proteins (58, 59) and is mediated by a tyrosine-containing motif. If a similar pH-dependent sorting process would operate on a chloride channel involved in intracompartmental acidification, it would reside in the compartment to be acidified until the correct pH is reached and would then be sorted to the Golgi. However, if the phenylalanine-containing motif can truly mediate localization of Gef1p to the Golgi, this invokes a different role for this type of motif than described for many other proteins.
The ER localization of many mutants may reflect a retention of misfolded protein by a quality control mechanism, but our experiments with concatamers and the effect of point mutations at the NH2 terminus argue against this possibility. Alternatively, forward sorting of Gef1p out of the ER may have been disrupted. For the yeast amino acid permeases a protein required for ER exit is known (60). A sorting signal promoting the ER exit of the vesicular stomatitis virus glycoprotein has been characterized in mammalian cells (61). Thus, it is conceivable that some of the mutants are no longer recognized as cargo destined for ER exit.
Our systematic alanine scanning of the COOH terminus showed that a
broad region of about 25 amino acids is also necessary for proper
function. This region coincides with the second CBS domain of Gef1p.
These domains, dubbed CBS for
cystathionine--synthase, have
recently been identified in a number of otherwise unrelated proteins
and occur in organisms as diverse as archaebacteria and man (43, 44).
The CBS domain is thought to fold into a compact structure of three
-strands with two short
-helices occurring after strands 1 and 3 (Fig. 4B). The function of these domains is presently
unknown. There are two CBS domains in the COOH termini of all
eukaryotic CLC proteins. Truncation of the muscle channel ClC-1 before
the second CBS domain led to a loss of function, and co-expression of
the truncated channel protein together with the COOH terminus
containing CBS2 again yielded functional channels (62). With ClC-0, a
similarly truncated channel could also be rescued by injecting the
COOH-terminal peptide encompassing CBS2 into the oocytes (63). This
effect could be blocked by brefeldin A, suggesting (in line with this
work) an effect of CBS2 on trafficking. In the baculovirus system,
however, an in-frame deletion of CBS2 in ClC-1 (leaving the extreme
COOH terminus intact) still gave currents, showing that CBS2 is not
absolutely necessary for ClC-1 function upon overexpression (64).
We have functionally delineated the borders of the second CBS domain, which coincide well with the borders suggested by Bateman (43) and Ponting (44). CBS1 is also essential, and CBS2 functions independently of its localization within the protein. This suggests that it acts as an independent structure, probably by binding to another protein domain. Whether this domain is on the channel itself, or on a different protein involved, e.g. in determining the localization of Gef1p, are problems whose importance clearly extends beyond CLC channels.
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ACKNOWLEDGEMENTS |
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We thank Monika Wormuth and Barbara Merz for excellent technical assistance. The generosity of many people who have contributed strains, plasmids, and antisera was crucial for this work and we thank them for their support: Rowan Chapman, Robert Fuller, Mike Lewis, Susan Michaelis, Sean Munro, Elke Pratje, Stefan Jentsch, Marc Rose, and Carmela Sidrauski. We thank members of the Jentsch laboratory, Rowan Chapman, Lily Jan, Dan Minor, and Noa Zerangue for valuable discussions and critical reading of the manuscript. Special thanks to Lily Jan for making it possible that this work could be finished in her laboratory as well as Rowan Chapman for extremely helpful comments on the project.
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Note Added in Proof |
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After this work was accepted, Gaxiola et al. (Gaxiola, R. A., Yuan, D. S., Klausner, R. D., and Fink, G. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4046-4050) reported that Gef1p and the copper-ATPase Ccc2p co-localize and showed that Gef1p is necessary for copper loading of Fet3p, explaining the iron requirement of the gef1 strain.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to T. J. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Howard Hughes Medical Institute, UCSF, Third and
Parnassus Ave., San Francisco CA 94143-0725.
§ To whom correspondence should be addressed. Tel.: 49-40-4717-4741; Fax: 49-40-4717-4839; E-mail: Jentsch{at}plexus.uke.uni-hamburg.de.
1
The abbreviations used are: CLC, generic name
for a specific chloride channel gene family; ClC-X, member X of this
chloride channel family; CBS domain, conserved protein domain found,
e.g. in cystathione--synthase; ER, endoplasmic reticulum;
TGN, trans-Golgi network; FITC, fluorescein isothiocyanate;
GFP, green fluorescent protein; LIM, low iron medium; WT,
wild-type.
2 T. Breiderhoff, T. Friedrich, and T. J. Jentsch, unpublished data.
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