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J. Biol. Chem., Vol. 281, Issue 41, 31070-31078, October 13, 2006
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12
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From the
Institute for Biochemistry I and ||Center for Molecular Medicine, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany, the Laboratory for Electron Microscopy, Cell Biology Department, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands, and the ¶School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom
Received for publication, May 16, 2006 , and in revised form, July 11, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sorting signal and a major µ1-adaptin binding site in vitro. This study provides the first insight into the function of mammalian Crn7 protein in the Golgi complex. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The coronin family comprises two groups of evolutionary conserved WD-repeat proteins (2, 3). Short coronins are actin-binding proteins playing accessory roles in the regulation of the dynamics of the actin cytoskeleton. Several of these protein have been shown to interact with Arp2/3 complex and help regulate the nucleation dynamics of actin filaments (reviewed in Ref. 3). Database search reveals up to six short coronins in human genome, and several of them have been characterized previously (4-9). Two of the known long coronin proteins, Caenorhabditis elegans and Drosophila POD-1s, bind to actin as well (10, 11). Absence of the worm POD-1 protein leads to accumulation of large cytoplasmic membrane structures along with defects in eggshell formation indirectly suggesting an exocytosis-related function. In Drosophila dpod-1-mutant flies axonal pathfinding is severely impaired (11).
We have previously identified mammalian Crn7 as a novel coronin family member and POD-1 homologue and demonstrated that it is abundantly expressed in most mouse tissues, is developmentally regulated and distributed between the cytosol and the Golgi (see also Fig. 1A), but is not associated with actin filaments (12). Crn7 sequence analysis revealed two WD-repeat blocks characteristic for longer coronins, two copies of the coronin signature motif and the lack of coiled coil regions. The protein lacks predicted signal sequence or a signal sequence cleavage site, as well as transmembrane domains (12).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-adaptin were from Sigma and against Src from Calbiochem, polyclonal antibodies against 
-adaptin and against AP-3 were from Santa Cruz Biotechnology. GFP-VSVG(ts-O45) plasmid was from Dr. Jennifer Lippincott-Schwartz. Alexa-633-labeled CTxB was from Molecular Probes (Utrecht, the Netherlands). Fine chemicals were purchased from Sigma unless indicated otherwise. Short interfering 21-mer RNA oligonucleotides (siRNA)3 targeted against human Crn7 (designed and supplied by Qiagen): siRNA(1)123, siRNA(2)+520, siRNA(3)1452, siRNA(4)163, siRNA(5)2639, siRNA(6)2055, siRNA(7)+60, siRNA(8)2454. Lower indices indicate the starting positions of corresponding sequences in the Crn7 mRNA; oligonucleotides 2 and 7 targeted fragments of the 3'-region of Crn7 mRNA starting 520 and 60 bases after the last coding base, respectively. COG3 siRNA was from Dr. Anna Shestakova. "Fast forward" transfection of HeLa using HiPerFect reagent (Qiagen) was performed according to the manufacturer's protocol. For plasmid transfections, we used the Lipofectamin Plus system (Invitrogen) or FuGENE 6 reagent (Roche Applied Science) according to the manufacturers' guidelines. The following synthetic peptides, high performance liquid chromatography-purified, were used: CON (KVEKIGEGTYGVVYK) was from Jena Biosciences, Jena, Germany and YEVV (GKGERQLYCYEVVPQ), AEVA (GKGERQLYCAEVAPQ), YELL (GKGDTRVFLYELLPE), and AELA (GKGDTRVFLAELAPE) were all from JPT Peptide Technologies GmbH, Berlin, Germany.
Cell Culture, Transfection, and Immunofluorescence—HeLa and Vero cells were from ATCC. Cells were grown according to standard procedures (12). For immunofluorescence, cells were grown on coverslips to approximately 50% confluence, fixed with 3.7% paraformaldehyde, rinsed twice with 20 mM glycine, blocked with 0.045% fish gelatin in the presence of 0.2% saponin, and incubated with primary antibodies diluted in blocking buffer. 0.02% saponin was present at all washing steps. Primary antibodies were detected using Cy3-, Alexa-568-, or Alexa-488-conjugated secondary antibodies (Sigma and Molecular Probes).
For immunofluorescence, siRNA was used to transfect 50% confluent cells on 12-mm (5 nM RNA) or 25-mm (30 nM RNA) coverslips in all experiments. For electron microscopy, 3 nM siRNAs were used to transfect 90% confluent HeLa cells on 12-mm coverslips. For Western blotting, fresh cells were plated onto 6-well plates at 50% confluence and analyzed 48 and 72 h after transfection with siRNA. As controls, mock-transfected and scrambled RNA-transfected cells were used.
Electron Microscopy—Mock- and siRNA-transfected HeLa cells were fixed for 10 min at room temperature in 100 mM phosphate buffer containing 4% paraformaldehyde and stored at 4 °C in phosphate buffer containing 1% paraformaldehyde until further use. Cells were osmicated, dehydrated, and embedded as described above. Ultrathin sections were cut and counterstained with uranyl acetate and lead citrate.
Immunoprecipitation and Western Blotting—Immunoblotting was performed according to standard procedures. Anti-Crn7 antibody (hybridoma supernatant) was used at 1:5 and mouse monoclonal anti-actin antibody (Sigma) at 1:20,000. Immunoprecipitation using protein A-Sepharose (Amersham Biosciences) was performed essentially as described (15). Where indicated, cells were incubated with protein cross-linker DSS (2 mM, Pierce) for 30 min at 20 °C prior to lysis.
Cloning, Expression, and Purification of AP µ-Chains—The coding sequences of truncated versions of µ1 (residues 158-423), µ2 (residues 158-435), and µ3 (residues 166-418) containing 5' NdeI and 3' EcoRI restriction sites were generated by PCR and cloned in frame into the vector pET28b to create a C-terminal fusion with a hexahistidine (His6) tag. The recombinant plasmids were used to transform Escherichia coli strain BL21, and protein production was induced by addition of isopropyl 
-D-thiogalactopyranoside for 3 h at 30 °C. The purification of µ-chain proteins was performed according to a standard protocol (Qiagen) using nickel-nitrilotriacetic acid-agarose as an affinity matrix. The purity of the µ-chain proteins, which were stored in 10 mM HEPES-KOH, pH 7.4, 500 mM NaCl, 10 mM -mercaptoethanol was controlled by SDS-PAGE, analytical gel filtration chromatography, and CD spectroscopy. Before any BIAcore experiment, the proteins were centrifuged for 30 min at 100,000 x g to ensure the removal of any possible protein aggregates.
Surface Plasmon Resonance-based in Vitro Binding Assays—The binding of recombinant His-tagged AP complex medium chains (µ1, µ2, µ3) to the putative Crn7 sorting signal-containing peptides and mutants thereof (see above) was measured by surface plasmon resonance using a BIAcore 3000 biosensor. All peptides were synthesized as 15-mers harboring a typical YXX motif close to their carboxyl terminus. In the mutant peptides, the critical tyrosine residue and the hydrophobic residue in the +3 position were substituted for alanine (for sequences see "Reagents"). Throughout the manuscript the peptides are named by their original or mutated tyrosine sorting motif. The peptides were immobilized via amino coupling onto carboxymethylated dextran (CM5) sensor surfaces according to the manufacturer's instructions. To minimize mass transfer effects, the amount of immobilized peptide was kept low (
200 resonance unit). After the peptide immobilization, the surface was regenerated with a pulse injection of 50 mM NaOH to remove non-covalently bound peptide. The AP complex µ-chains were injected in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Polysorbate 20; from BIAcore) followed by regeneration. The obtained sensorgrams were used to calculate the kinetic rate constants for association (Ka), dissociation (Kd), and the equilibrium rate constants (KD). µ-Chain binding to Crn7 peptides could be best modeled assuming a 1:1 interaction which is consistant with the structural information on peptide binding to AP µ-chains (16, 17) and several other published reports using the same technology (18, 19).
VSV Envelope G Glycoprotein (VSVG) and CTxB Trafficking Assays—Trafficking assays were performed essentially as described (20, 21). In brief, for the VSVG trafficking assay, HeLa or Vero cells were co-transfected with GFP-VSVG-tsO45 and CFP-GalT plasmids, kept at 37 °C for 2 h, and at 40 °C for 16 h. Cells were then shifted to 32 °C in the presence of 150 µg ml-1 cycloheximide in phenol red-free Dulbecco's modified Eagle's medium high glucose medium supplemented with 10% serum and 20 mM HEPES to allow the synchronous export of VSVG from the ER. Where indicated, cells were transfected with siRNA 16 h prior to transfection with VSVG. For the CTxB trafficking assay, Alexa-633-labeled CTxB (500 ng ml-1) was added to the cell culture medium for 3 min at room temperature, followed by washing, internalization at 37 °C, fixation, and immunolabeling.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Defects in Anterograde Transport in Crn7 KD Cells—The dramatic defects we observed in Crn7 knockdown cells prompted us to address the question whether these defects correlate with impairment of Golgi transport. We monitored the Golgi export of the VSVG, known to hijack the anterograde transport system to reach the cell surface after proceeding through the ER and the Golgi. The experiments were performed with a mutant VSVG (VSVG-tsO45) carrying a single point mutation F204S (22), which renders it temperature-sensitive with regard to its intracellular trafficking. The mutant protein accumulates in the ER at 39.5 °C and is released to the Golgi complex upon a temperature switch to 32 °C (see "Materials and Methods"). This mutant is widely used to study the dynamics of protein trafficking along the biosynthetic pathway (20, 23).
In mock-transfected HeLa cells, VSVG-tsO45-GFP gradually accumulated in the Golgi complex upon shifting the temperature to 32 °C (data not shown). Starting at 10 min after the shift, formation of characteristic tubular compartments originating from the Golgi was observed. These intermediates, morphologically similar to Golgi-to-plasma (GPC) membrane carrier precursors described previously (24), released vesicles targeted to the plasma membrane (Fig. 2A). Apart from vesicles derived from GPC precursor-like tubules, we observed direct formation of vesicular transport intermediates leaving the Golgi in the direction toward the plasma membrane.
Cells treated with Crn7 RNAi and recognized as Crn7 knock-down by their scattered CFP-GalT (Golgi galactosyltransferase) pattern were imaged under the same conditions and demon-strated a similar rate of accumulation of VSVG in the Golgi apparatus (data not shown). However, we did not observe any release of VSVG in GPC precursor-like tubules or vesicles from the Golgi even 120 min after the temperature shift. At all times, the VSVG GFP signal was present in the scattered Golgi remnants (Fig. 2B). We further quantified the VSVG GFP signal intensity at the plasma membrane in 100 images spanning 30 min of time in mock-transfected and Crn7 RNAi cells using MetaMorph image analysis software. In control cells, the GFP fluorescence intensity gradually increased to nearly double the initial value, reflecting the delivery of VSVG from the Golgi complex, whereas in RNAi cells, the GFP fluorescence signal at the plasma membrane remains at basic threshold level, which is approximately 50% of that of mock-transfected cells (Fig. 2E).
To confirm our data on VSVG export defect, we assessed VSVG trafficking in Crn7 KD Vero cells, a primate kidney cell line. We did not observe any GPC-like structures in either control or KD Vero cells (Fig. 2, C and D). However, vesicular transport of VSVG from the Golgi to cell surface was greatly reduced in Crn7 KD cells as compared with the control (Fig. 2D, see also supplemental Movies 1 and 2). Note that in Vero cells, Crn7 knockdown led to the formation of significantly larger, compacted Golgi remnants than in HeLa cells (compare Fig. 2, B and D). Quantification of cell surface VSVG further confirmed the defect in VSVG delivery to cell surface comparable with that in HeLa cells (data not shown).
The Golgi does not only export trafficking intermediates but also receives material by retrograde trafficking. To test whether the retrograde transport to the Golgi requires Crn7, we studied the intracellular dynamics of CTxB, the non-toxic B-subunit of the AB5 toxin, cholera toxin (25). In control cells, Alexa-633-labeled CTxB (CTxB-633) was internalized by cells and reached the Golgi complex approximately 30 min after its addition to the cells (Fig. 2G), which is in good agreement with data from the literature (13, 21). In Crn7 RNAi cells (asterisk in Fig. 2, F and G), CTxB-633 reached the scattered perinuclear Golgi remnants present in these cells with kinetics indistinguishable from those observed for its arrival in the Golgi complex in control cells (Fig. 2G). Thus, the protein import into the trans-Golgi network (TGN) is Crn7-independent.
The Golgi export block that we detected in Crn7 siRNA cells can be due to numerous reasons, such as defects in proper cargo protein modification and/or machinery for protein sorting and recruitment. To experimentally assess the possibility of interplay of Crn7 with cargo glycosylation, we used LAMP2, a cargo protein destined to late endosomes and lysosomes, in Crn7 siRNA and control cells. In Crn7 KD cells, the fully glycosylated form of LAMP2 appeared at approximately 110 kDa, and a band of similar size was detected in control siRNA-transfected cells (Fig. 2H). As a positive control, we used siRNA directed against the COG3 protein, a part of the multisubunit Golgi protein complex acting at several stages of glycoprotein and glycolipid metabolism (Ref. 26 and references therein). COG3 knock-down is correlates with a massive defect in Golgi glycosylation (26). In accordance with literature data, immature LAMP2 migrated as a protein of lower molecular mass at approx. 80 kDa in COG3 RNAi but not Crn7 RNAi cells. Thus, Crn7 knockdown influences the Golgi export of cargoes but not cargo glycosylation.
Interaction of Crn7 with the Clathrin Adaptor AP-1—We further wanted to test whether Crn7 knockdown affects cargo selection and export vesicle formation. Adaptor proteins such as monomeric GGAs and heterotetrameric adaptor complexes (AP) are the key factors for the incorporation of cargo membrane proteins into nascent coated vesicles (1, 27). AP complex subunits are called adaptins. Binding of the membrane proteins to adaptor proteins is mediated by small peptide sequences (sorting signals), localized in the cytosolic portions of the proteins, such as tyrosine-based signals of the YXX type (X, any residue; , a bulky hydrophobic residue). The µ-subunits of AP complex tetramers interact with YXX motifs of cargo proteins, while -subunits interact with clathrin (1). There are four AP complexes in mammalian cells (AP-1 to AP-4), all of them functioning in the same way but at distinct cellular sites. While AP-2 is restricted to the plasma membrane and is involved in clathrin-coated vesicle formation during endocytosis, AP-1 has the same function in the TGN and most likely in endosomes. The functions of AP-3 and AP-4 are less well understood (1).
Crn7 harbors two sequences matching a classical YXX motif localized directly downstream of each coronin core domain (Fig. 3A). We tested whether these motifs of Crn7 can bind to purified µ-subunits of the AP-1,-2, and-3 adaptor complexes. 15-mer synthetic peptides harboring each of the YXX motifs close to their carboxyl terminus were used along with mutant peptides where the tyrosines and the hydrophobic () amino acids in the +3 positions were replaced by alanine residues. The peptides were covalently coupled via their amino terminus to a CM5 surface of a BIAcore 3000 biosensor. Sub-sequently, purified recombinant µ1, µ2, or µ3 subunits were injected at different concentrations. The peptide comprising Tyr288 bound with high affinity to µ1 and µ2 but not to µ3 (Fig. 3B, YEVV). Binding was specific as indicated by the low degree of background binding observed for the mutant peptide. The second more distal Crn7 peptide containing Tyr758 also bound to µ1 and µ2 but only at a very low level (Fig. 3B, YELL). Although the evaluation of the data for the Tyr288 peptide binding to µ1 and µ2 did not perfectly match the expected model for a one-to-one type of interaction, we could estimate the in vitro equilibrium binding rate constant KD at 150 nM, which fits well to published data on signal binding to µ-chains (28). Binding of both µ1 and µ2 subunits was concentration-dependent and reversible, with minimal re-binding during the dissociation phase (Fig. 3, C and D). Fig. 3E shows the purity of µ-subunit preparations.
To ascertain that the Crn7 binding to AP-1 and AP-2 is significant in vivo, we tested whether Crn7 can be co-immunoprecipitated with AP complexes. As the YEVV motif was shown to interact with µ1 and µ2 in vitro, we performed immunoprecipitation experiments using antibodies recognizing the complexspecific subunits of AP-1,-2, and-3. Crn7 was detectable in AP-1 pull-down, but not AP-2 or AP-3 pull-downs (Fig. 4A), inferring an in vivo interaction of Crn7 with AP-1 but not with AP-2 or -3. This observation was confirmed by reverse immunoprecipitation of AP-1 together with Crn7 from cell lysate derived from HeLa cells treated with protein cross-linker DSS (Fig. 4A, lower panel).
Crn7 co-localizes with AP-1 on the Golgi but not on AP-1-positive endosomes (Fig. 4B), suggesting that the interaction of Crn7 and AP-1 takes place at Golgi membranes. Taken together, our in vitro binding and immunoprecipitation data suggest that Crn7 interacts with AP-1.
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motif of Crn7 as demonstrated by surface plasmon resonance experiments, and it does not co-immunoprecipitate with Crn7 (Fig. 4A). This important observation strongly infers that the presence of a putative sorting signal in the protein sequence is not sufficient for the interaction with any given sorting machinery. Specificity of such interaction may be regulated by amino acid composition in the vicinity of the sorting signal or by additional protein factors.
Localization of AP-1 and AP-1-dependent Cargo in Crn7 KD Cells—Next, we were interested to know whether Crn7 acts upstream or downstream of AP-1 in the Golgi export pathway and whether Crn7 is required for the targeting of AP-1 to the Golgi. Subcellular distribution of AP-1 in control and Crn7 KD cells was studied by immunofluorescence microscopy using an anti-body specific to -adaptin (Fig. 4C, upper panel). In both control and RNAi cells, AP-1 was detectable on Golgi membranes and endosomes. Crn7 KD cells, however, displayed marked increase of -adaptin on the scattered Golgi. Thus, Crn7 knock-down appears not to interfere with the ability of AP-1 to be recruited to the cargo, and Crn7 possibly acts downstream of AP-1 recruitment.
VSVG export from the Golgi has never been demonstrated to depend on AP-1. As we have established the interaction of Crn7 with AP-1 in vivo and in vitro, we were interested whether bona fide AP-1 cargoes require Crn7 to be exported from the Golgi complex. To this end, we studied the distribution of 46-kDa mannose 6-phosphate receptor MPR46 in HeLa cells treated with Crn7 siRNA. In Crn7 KD cells, MPR46 was retained and markedly accumulated in the scattered Golgi (Fig. 4C, lower panel). A lysosomal marker protein LAMP2 showed similar behavior (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Crn7 is the first coronin found to be localized to the Golgi and to directly execute a membrane trafficking-related function. Although mutant phenotypes of both worm and fly homologues (see Introduction) may be explained by defects in Golgi function and anterograde trafficking, we do not envision the same function for Crn7 and either of the two POD-1 proteins. We have no data implying any interaction of Crn7 with the cytoskeleton, while there is no indication for the Golgi localization of any of the POD-1 proteins. We anticipate the Golgi localization and Golgi-related function to be unique for vertebrate or mammalian POD-1 homologues. This consideration is in line with the fact that Tyr288 and Tyr758, parts of the two putative tyrosine-based sorting signals in Crn7, are not present in the C. elegans POD-1 protein, while in Drosophila, a tyrosine residue is present in a vicinity of Tyr288 position, but its amino acid environment is very different, and so is the predicted tertiary structure in this area.5 We suggest that the specificity of functions of POD-1/Crn7 subfamily of proteins is specified by unique non-core subdomains rather than conserved WD-repeat-containing core regions.
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AP-1 to Golgi membranes, as the amount of AP-1 on Golgi in Crn7 KD cells is enriched rather than down-regulated. Most probably, the accumulation of AP-1 on Golgi membranes reflects the fact that the cargo stuck in the compartment is already primed for export, e.g. the complex between cargo and AP-1 has already formed, but the formation of transport intermediates is impaired.
The marked accumulation of AP-1 on the Golgi together with accumulated cargo would suggest that the role of Crn7 in the Golgi export may be confined to the events downstream of the cargo-adaptor interaction. Such events include the generation of membrane curvature followed by membrane fission. Another such event may be interaction with (hypothetical) non-clathrin, non-COPI/II coat proteins.
Several proteins have been implicated in the membrane fission reaction in the TGN. One of the most intriguing is protein kinase D (PKD), an enzyme localizing to the TGN by means of interaction with diacylglycerol and required for the formation of post-Golgi transport intermediates (29-32). The role of PKD can be precisely attributed to membrane fission as the overexpression of kinase-inactive PKD leads to the formation of long TGN-derived tubes containing cargo but not resident enzymes or coat proteins (29, 32). Our data allow us to speculate that Crn7 acts upstream of PKD in the secretory pathway as Crn7 KD does not result in the formation of stabile cargo-containing tubules arrested in fission.
It remains to be established how exactly Crn7 is targeted to the Golgi, what other proteins it interacts with, and what the role of the cytosolic pool may be. Another important question arises from our earlier observation that Crn7 is tyrosine-phosphorylated on membranes (12) but not in the cytosol. Further studies will be required to find out whether phosphorylation is required for Crn7 targeting to Golgi membranes or for its function and what kinase(s) participate in this event.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Movies 1 and 2.
1 Authors are recipients of fellowships from Graduate Schools in Genetics and Functional Genomics, University of Cologne, and in Behavioral and Cognitive Neurosciences, University of Groningen, respectively.
2 To whom correspondence should be addressed: Division of Biological Sciences, University of California San Diego, 9500 Gilman Dr., MC0634, La Jolla, CA 92093. E-mail: vrybakin{at}ucsd.edu.
3 The abbreviations used are: siRNA, small interfering RNA; DSS, 2,2-dimeth-yl-2-silapentanesulfonic acid; ER, endoplasmic reticulum; GFP, green fluorescent protein; VSVG, VSV envelope G glycoprotein; RNAi, RNA interference; KD, knockdown; GPC, Golgi-to-plasma; TGN, trans-Golgi network; PKD, protein kinase D.
4 A. A. Noegel, unpublished data.
5 V. Rybakin, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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