Antibodies and Reagents
Antibodies against WIPI49 were raised in rabbits using a peptide corresponding to its C-terminal 20 amino acids (NEFPPIILCRGNQKGKTKQS), which had been conjugated to keyhole limpet hemocyanin (Calbiochem, La Jolla, CA). Sera were immunopurified using standard procedures after the peptide had been conjugated to actigel resin (Sterogene Bioseparations, Arcadia, CA). Other antibodies used in this study were murine anti-GM130 (Transduction Laboratories, Lexington, KY), murine anti-p230 (Transduction Laboratories), murine antigamma adaptin (Transduction Laboratories), goat anti-EEA1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-CI MPR (Gift of Dr Sharon Tooze), monoclonal 9E10 antimyc and monoclonal 3E1 anti-GFP (Central Services, CRUK, London).
Cloning and Expression of WIPI49
A full-length ORF for WIPI49 was amplified from IMAGE clone 752767 by PCR using the 5′ primer WIPI49f, which contained a HindIII site, (GGAGGCAAGCTTCGATGGACGCTCCCCCGGGC) in combination with the 3′ primer WIPI49r, which contained an EcoRI restriction site, (GTGTGCGAATTCAGTCATGACTGCTTCGTTTTGCC) (bold text refers to incorporated restriction site throughout). The resultant PCR product was cloned into pCR-Script (Stratagene, La Jolla, CA). After sequencing to confirm fidelity of amplification, the WIPI49 ORF was spliced into pEGFP-C1 and pDsRED2-C1 vectors (CLONTECH, Palo Alto, CA) by restriction digest with EcoRI and HindIII. A Myc-tagged version of WIPI49 was constructed by amplification of WIPI49 by the 5′ primer MycWIPI49f, which contained an EcoRI site (CTCGAATTCGCGATGGACGCTCCCCCGGGCGGG) and the 3′ primer MycWIPI49r, which contained a HindIII site (GGTAAGCTTTGACTGCTTCGTTTTGCCCTTC), and the amplified product was cloned into pCR-Script, sequenced, and subsequently inserted into pcDNA3.1-Myc by restriction digest with EcoRI and HindIII. To express WIPI49 in mammalian cells as a GST fusion the WIPI49 ORF was amplified by PCR using the 5′ primer WIPIGSTf, which contained a PstI site (TCGAGCCTGCAGTTATGGACGCTCCCCCGGGCG) in conjunction with the 3′ primer WIPIGSTr, which contained a NotI site (TGCAGAGCGGCCGCGACTGCTTCGTTTTGCCC). The PX domain of Snx3 was amplified using the primer combination gSnx3Pxf (GACGCCTACCTGCAGATGAGCAACTTCCTCGAGATCGACG) and gSnx3Pxr (CGGACCGTGCGGCCGCGCATGTCTTATTTTAGATGGAG).
Postamplification the PCR product was cloned into pCR-Script, sequenced to confirm fidelity and subsequently spliced into pMT2-SM-GST by restriction digest with the enzyme combination NotI and PstI.
The WIPI49 R226A, R227A mutant was generated by megaprimer mutagenesis PCR. The megaprimer was generated by amplification of the first 680 bases of NP_060453 with the 5′ primer 49f (CGAGCTCAAGCTTCGATGGACGCTCCCCCGGGCG) and the 3′ primer 49RRr (CACATACCTTTTCATCGCTGCAGCGGCCTCATAGAGCTTTTGCCC, mutated bases shown in bold). The amplified product was gel-purified and used in a second round of PCR in combination with 49r (ACTGCAGAATTCTCATGACTGCTTCGTTTTGCC). The resulting product (49RR) was digested with HindIII and EcoRI before splicing into pEGFP or pDsRED2. For generation of the GST-fusion vectors, 49RR was amplified using the primer combination WIPIGSTf and WIPIGSTr (see above), and the product was inserted into pMT2-GST by restriction digest with NotI and PstI.
Cell Culture and Transfection
African Green monkey (Cos7) cells were grown in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 μg/ml penicillin, 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 10% CO2. Cells were transiently transfected with plasmids of interest using LIPOfectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Cells were typically analyzed 36 h posttransfection.
Sequences were aligned using ClustalX and alignments saved in GCG-MSF format before preparation for presentation in MacBoxShade. Phylogenetic trees were prepared by bootstrapping in ClustalX and presented using NJPlot. Identity/similarity matrices were generated using MacBoxShade (www.ISREC.ISB-S1B.CH/ftp-server/boxshade/macboxshade/
). Structural predictions were determined using the 3D-PSSM at URL: www.sbg.bio.ic.ac.uk/3dpssm/
For the inhibition of PtdIns3K cells were serum-starved for 1 h before treating with 10 μM LY294002 for the times indicated. To disrupt the microtubule cytoskeleton, cells were treated with 10 μg/ml nocodoazole for the times indicated.
Cells seeded on glass coverslips were washed with PBS before fixing in 4% PFA for 10 min at room temperature. After fixation cells were rinsed with PBS, permeabilized for 5 min in 0.2% Triton X-100, rinsed with PBS, and blocked in 1% BSA for 30 min. Cells were subsequently incubated with primary antibody for 2 h, washed four times with PBS, incubated with the relevant secondary antibodies for 1 h, washed three times with PBS and once with water, and finally mounted in Mowiol. 4-88 (Calbiochem). Slides were examined using a confocal laser scanning microscope (Axioplan2 with LSM510; Carl Zeiss, Thornwood, NY) equipped with a 63×/1.4 plan-APOCHROMAT oil immersion objective.
Live Cell Time-Lapse Imaging
For live cell imaging cells were grown on 35-mm glass-bottom microwell dishes (Matec, Northborough, MA). Imaging was performed in an environmental chamber at 37°C supplemented with 5% CO2. Cells were maintained in phenol red-free DMEM containing 10% FBS. Cells were examined 20-24 h posttransfection using an Olympus IX70 microscope fitted with a Wallac UltraVIEW Confocal Scanner (Perkin Elmer-Cetus, Boston, MA), an E-662 LYPZT Amplifier Pieza Disk using the UltraVIEW Imaging Suite software package in Temporal Module setting (Perkin Elmer-Cetus). DsRED2 chimeric proteins were excited at 565 nm and detected at an emission wavelength of 583 nm, whereas GFP chimeras were excited at 488 nm and detected with a split emission spectrum of 525/550 nm. Quicktime movies were constructed from sets of sequential TIFFs using the AQM 2001 Kinetic Acquisition Manager software (Kinetic Imaging Ltd., Liverpool, United Kingdom).
Purification of GST-fusion Proteins
GST-fusion proteins were expressed in Cos7 cells transfected with pMT2-SM constructs containing the ORF of interest. Cells were allowed to express the fusion protein for 36 h after transfection before harvest in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.5% NP40). Lysates were clarified by centrifugation at 25,200 × g for 10 min at 4°C before addition to GSH-Sepharose beads (Amersham, Amersham, UK), which had been washed previously in lysis buffer. GST-fusion proteins were incubated with beads overnight at 4°C with tumbling on a rotary wheel. After binding, beads were washed five times with 10 volumes of lysis buffer, and GST-fusion protein was eluted with 10 mM reduced GSH, 50 mM Tris-HCl, pH 8.0.
Liposomes were prepared according to a protocol based on that of Cozier et al.
). Briefly, lipid mixtures of brain-derived phosphatidylethanolamine (Avanti, Birmingham, AL), synthetic dioleyl-phosphatidylserine (Avanti) and synthetic 1-palmitoyl-2-oleyl-phosphatidylcholine (Avanti) supplemented with specific diC16 phosphoinositides (Cell Signaling Technology, Beverly, MA) were dried under a stream of nitrogen in glass tubes to form a film of lipid. A 10× lipid stock buffer (20 mM KCl, 20 mM HEPES, pH 7.4, 0.2 mM EDTA) was added to the dried lipids and liposomes generated in a bath sonicator adaptor of a Branson Sonifier 250 sonicator (Danbury, CT). Aggregated material was removed by centrifugation of lipids in an Eppendorf centrifuge at 14,000 rpm for 10 min before dilution of liposomes into a 1× reaction buffer (0.12 M NaCl, 0.1 mM EGTA, 0.2 mM CaCl2
, 1.5 mM MgCl2
, 1 mM DTT, 5 mM KCl, 20 mM HEPES, pH 7.4, 1 mg/ml ovalbumin). Protein (250-500 ng) was incubated with liposomes for 10 min at room temperature before centrifugation at 100,000 × g
for 30 min in a TL100 bench-top ultracentrifuge. Supernatants were removed and pelleted material resolved by SDS-PAGE.
Biosynthetic Labeling of Cathepsin D
Cathepsin D was labeled with [35S]methionine (Amersham) essentially as previously described (Davidson, 1995). Briefly, transfected Cos7 cells grown on 35 × 10-mm Petri dishes were starved for 3 h in DMEM lacking methionine, supplemented with 10% FCS, which had been dialyzed, for 3 h. Cells were then labeled with 125 μCi [35S]methionine for 30 min. Postlabeling cells were washed twice with DMEM/10%FCS and then chased for 3 h in normal media. Subsequent to chasing, cell media was collected and reserved, and cells were washed twice in PBS and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 0.5% NP40, mini-complete protease inhibitors; Sigma, St. Louis, MO) and clarified by centrifugation for 10 min. Then equivalent amounts of cellular media and cell lysates were precleared with protein A before immunoprecipitation with monoclonal anti-cathepsin D (Serotec, Oxford, UK) at 4°C overnight. Postprecipitation antibody-protein conjugates were harvested using protein A-Sepharose beads, and beads then were washed extensively in lysis buffer before resolution of immunoprecipiates by SDS-PAGE. After electrophoresis gels were stained with Coomassie, treated with ENHANCE (New England Nuclear, Boston, MA), dried down, and then exposed to a phosphoimager.
Isolation of Clathrin-coated Vesicles from Rat Brain
Ten rat brains were washed in buffer 1 (25 mM HEPES, pH 7.4; 125 mM potassium acetate; 5 mM magnesium acetate; 1 mM DTT), resuspended to a volume of 40 ml and homogenized with 20 strokes of a Potter homogenizer. Homogenates were spun at 8000 × g in an SS34 rotor for 20 min, and supernatant was collected and ultracentrifuged at 150,000 × g for 40 min in a 70Ti rotor. The pellet was resuspended in 10 ml of buffer 1, homogenized, and added to an equal volume of buffer 1 containing 12.5% Ficoll and 12.5% sucrose. The resultant solution was spun in a 70Ti rotor at 46,000 × g for 20 min, and the supernatant was diluted 1:5 in buffer 1 and ultracentrifuged at 95,000 × g for 60 min in a 45Ti rotor. The pellet was homogenized in 15 ml of buffer 1 and left on ice for 1 h after which it was spun at 16,000 × g for 10 min. Finally the supernatant was spun at 80,000 × g in an SW40 rotor for 2 h to pellet CCVs.
Preparation of Cos7 Cell Lysate Fractions
For examination of endosomal densities, Cos7 cells were scraped and washed in PBS before resuspension in homogenization buffer (HB; 0.25 M sucrose, 0.5 mM EDTA). Cells were burst by passing through a Cellcracker (HGM) and a postnuclear supernatant (PNS) prepared. The PNS was layered onto a continuous sucrose gradient ranging from 21% sucrose to 54% sucrose and centrifuged in a SW40 rotor at 87,000 × g overnight. After centrifugation 24 fractions were collected and each resolved by SDS-PAGE.
RNAi was conducted using the pSUPERpuro system (OligoEngine, Seattle, WA). Three specific 23-base long regions were selected from the DNA sequence of WIPI49. These were incorporated into a synthetic 64mer oligonucleotide designed for hairpin RNA expression. Both sense and antisense oligonucleotides were made. The three pairs of oligonucleotide were annealed to each other and subsequently inserted into pSUPERpuro. The primer combinations used are as follows: for pSUPERpuro-WIPI49I, RNAiWIPI49f2 g a t c c c c g t t a t t c c t g a a c a t g a g t t t c a a g a g a a c t c a t g t t c a g g a a t a a c t t t t t g g a a a and RNAiWIPI49r2 a g c t t t t c c a a a a a g t t a t t c c t g a a c a t g a g t t c t c t t g a a a c t c a t g t t c a g g a a t a a c g g g, for pSUPERpuro-WIPI49II, RNAiWIPI49r8 a g c t t t t c c a a a a a g g t a t g t g a c aatcagctctctcttgaagagctgattgtcacataccgggand RNAiWIPI49f8 ga t c c c c g g t a t g t g a c a a t c a g c t c t t c a a g a g a g a g c t g a t t g t c a c a t a c c t t t t t g g a a a, for pSUPERpuro-WIPI49III, RNAiWIPI49f13 g a t c c c c g c c t g a c t t c a g g g g a g a t t t c a a g a g a a t c t c c c c t g a a g t c a g g c t t t t t g g a a a and RNAiWIPI49r13 a g c t t t t c c a a a a a g c c t g a c t t c a g g g g a g a t t c t c t t g a a a t c t c c c c t g a a g t c a g g c g g g. After confirmation of the constructs Cos7 cells were transfected with the appropriate plasmid, and transformants were selected for in media containing 2.5 μg/ml puromycin.