The mAb AP.6 (Affinity Bioreagents, Golden, CO) was used to detect the α-adaptin subunit of AP-2 (Chin et al., 1989
), and TD.1 (Covance Research Products, Denver, PA) was used to identify clathrin heavy chain (Nathke et al., 1992
). Fluorescein isothiocyanate (FITC)-conjugated EEA1 polyclonal antibodies were obtained from BD Biosciences (San Jose, CA). AAK1L isoform-specific polyclonal antisera (rabbit 273) against a peptide containing the last 17 amino acids of AAK1L (LPNLARSLLLVDQLIDL) conjugated to KLH was commercially produced (Sigma Genosys, St. Louis, MO). Polyclonal antisera against the AAK1 ΔAID region (rabbit 6370) were previously generated and described (Conner and Schmid, 2003b
). The mAb E7 that recognizes beta-tubulin was a generous gift from Dr. Jeff Miller (University of Minnesota).
Clathrin and Adaptor Protein Isolation
The extended 3′ AAK1L fragment was isolated using a nested PCR approach from a random-primed human brain cDNA library (EMD Biosciences, San Diego, CA). The first round of PCR primers included an AAK1 gene–specific primer (5′-ATTCAAGCCCCAGTGAGACAAC-3′) and a vector primer (5′-GGTTATGCTAGTTATTGCTCAGC-3′). The product of this reaction was used for a second round of PCR using two nested gene-specific primers: 5′-AGCCAAAAAGTTCAGACCACTC-3′ and 5′-CTATAGGAGAAGGAAAGGGGT-3′. Full-length AAK1L was then generated by PCR using primers 5′-GAATTCGATGAAGAAGTTTTTCGACTCCCGG-3′ and 5′-GAATTCCAGGTCTATGAGCTGATCCA-3′ both of which incorporated an EcoRI site that was used for subsequent subcloning into baculovirus vectors. PCR products were subcloned into pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced.
Recombinant Protein Production
Full-length AAK1L fusions proteins were generated by subcloning the AAK1L EcoRI fragment from pCR2.1 into the pFastbac1 vector (Invitrogen), which had been previously engineered to contain an in-frame carboxy terminal glutathione S-transferase (GST) or 6HIS affinity tag. The pFastbac1 plasmid was then used to generate baculovirus and subsequently used for protein expression in SF9 cells using the Bac-to-Bac System according to manufacturer protocols (Invitrogen). An AAKL PCR product using primers 5′-GAATTCCAAAGCTGATGTTGCTGTTGAGAGTCTC-3′ and 5′-CTATAGGAGAAGGAAAGGGGT-3′ was subcloned from pCR2.1 into the EcoRI site of pGEX4T-3 (Amersham Pharmacia, Piscataway, NJ) to generate a CBD2-GST fusion construct for bacterial expression in the Rosetta strain (EMD Biosciences).
Adenoviruses encoding an amino terminal myc-tag fused to AAK1L (full-length or CBD2) was generated by subcloning the AAK1L insert from pCR2.1 (SpeI/NotI sites from pCR2.1 used) into the SpeI/NotI site of pADTet7. Short hairpin RNA (shRNA)-encoded adenovirus was constructed by shuttling the H1 histone promoter cassette from pSuper (OligoEngine) into the XhoI site of the pADTet7 plasmid backbone. Hairpin-encoding primers specific for AAK1 were generated (sense: 5′-GATCCCCGTGCTCATTCTGATGGACTTTCAAGAGAAGTCCATC-AGAATGAGCACTTTTTGGAAA-3′; antisense: 5′-AGCTTTTCCAAAAAGTG-CTCATTCTGATGGACTTCTCTTGAAAGTCCATCAGAATGAGCACGGG-3′)and cloned into the BglII/HindIII site of pSuper according to manufacturer protocols before shuttle into pADTet7. The construct was sequence verified before use. The pADTet7 plasmids were then used for adenovirus production as previously described (Damke et al., 1995
Northern Blot Analysis
Human polyA+ RNA tissue blots (Origene, Rockville, MD) were probed with antisense RNA probes for AAK1 using the Strip-EZ RNA kit (Ambion, Austin, TX) and hybridized according to manufacturer protocols. AAK1 antisense RNA probes were generated using AAK1 cDNA in pCR2.1-containing nucleotides 1777–2466, a region present in both AAK1s and AAK1L isoforms. An AAK1L-specific antisense RNA probe was generated from a pCR2.1 construct containing AAK1L nucleotides 2468–2880. As a control for mRNA loading, a DNA probe was generated using human β-actin cDNA using the DECAprime II kit (Ambion, Austin, TX) according to manufacturer's protocols. All probes were 32P-labeled and hybridized probes were detected by exposing blots to a phophorimaging screen (Amersham Pharmacia) and detected with a Storm scanner (Molecular Dynamics, Sunnyvale, CA).
Affinity matrices were generated essentially as described (Conner et al., 2003
). Full-length or truncated AAK1 GST fusion proteins immobilized on glutathione-Sepharose beads (Amersham Pharmacia) were incubated for 1 h at room temperature with clathrin triskelia or adaptor proteins from bovine brain. Matrices were then washed with 10 column volumes of phosphate-buffered saline-Tween (PBST) and bound protein was analyzed by SDS-PAGE followed by immunoblot analysis using adaptor protein or clathrin-specific antibodies.
Kinase assays were performed as described (Wilde and Brodsky, 1996
). Briefly, isolated baculovirus expressed GST fusions of AAK1s or AAK1L with or without adaptor proteins and/or clathrin were mixed in kinase buffer (150 mM KCl, 5 mM MgCl2
, 100 μM [γ-32
P]ATP) for 40 min at room temperature. The kinase reaction was stopped by the addition of 5× protein sample buffer and boiling for 2 min at 100°C. Proteins were resolved on a 10% polyacrylamide gel, dried, exposed to a phosphoimaging plate, and analyzed using the ImageQuant Software package (Molecular Dynamics). Endogenous kinase activities found in isolated adaptor protein preparations were inactivated by pretreatment with 1 mM 5′-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA, Sigma) for 60 min at room temperature. Unbound FSBA was removed from protein preparations by gel-filtration using G25 mini-spin columns (Amersham).
For immunofluorescence experiments, HeLa cells were grown on coverslips and then fixed with ice-cold acetone for 10 min followed by methanol extraction. Cells were washed three times with PBST and then incubated with the designated antibodies for 1 h at room temperature. Samples were washed three times with PBST and the appropriate secondary antibodies were added at 1:5000 dilution and incubated for 1 h at room temperature. Samples were visualized by epifluorescence with the appropriate filter set using a Zeiss Axioscop 2 microscope equipped with a Zeiss Axiocam MRm digital camera and 40× and/or 100× objectives. Images were captured using Zeiss Axiovision software release 3.1 (Carl Zeiss, Thornwood, NY).
Single-Round Transferrin Internalization Assay
Stably transformed Tetracycline transactivator (tTA) HeLa cells were cultured in DMEM supplemented with 10% fetal calf serum, 100 U/ml streptomycin/penicillin, and 400 μg/ml G418 as previously described (Sever et al., 2000
) and grown to ~70–80% confluency before use in internalization assays. Cells were detached from 100-mm dishes by the addition of 1.0 ml PBS/5 mM EDTA at room temperature for 5 min and transferred to 1.5-ml microcentrifuge tubes. Cells were gently pelleted by centrifugation at 1000 × g
, washed twice with 1.0 ml ice-cold PBS containing 1 mM MgCl2
, 1 mM CaCl2
, 0.2% bovine serum albumin (BSA), and 5 mM glucose (PBS4+
), and resuspended at 2 × 106
cells/ml in PBS4+
containing 5 μg/ml biotinylated transferrin (BXX-Tfn) and incubated 60 min on ice to allow ligand binding to the receptor. Cells were washed twice with ice-cold PBS4+
to remove unbound transferrin, resuspended at 2 × 106
cells/ml in PBS4+
, and split into 50-μl aliquots (1 × 105
cells). Samples were then transferred en masse to 37°C for the indicated times. Endocytosis and recycling were stopped by returning samples to ice. Internalized biotinylated transferrin was quantitated by ELISA assay as previously described (Carter et al., 1993
tTA HeLa cells were grown on glass coverslips to ~70–80% confluency. Before endosomal loading with labeled transferrin, cells were washed with PBS and serum-starved for 30 min at 37°C in DMEM supplemented with 0.5% BSA. Starvation medium was then replaced with prewarmed DMEM/0.5% BSA containing 5 μg/ml Alexa488-labeled transferrin (Invitrogen), and cells were incubated for 60 min at 37°C to allow accumulation of labeled transferrin in the endosomal compartment. After cell loading, a coverslip from each condition was removed, transferred to ice, fixed with 4% formaldehyde, and represented the T = 0 time point. The remaining slides were washed with PBS, the media was replaced with DMEM/0.5% BSA containing 50 μg/ml unlabeled transferrin prewarmed to 37°C and incubated at 37°C to allow recycling from the endosomal compartment back to the plasma membrane. After a 40-min incubation, coverslips were transferred to ice and fixed as described above.
To selectively load the early endosome, cells were processed as before except that the transferrin (biotinylated transferrin) internalization step occurred at 16°C for 60 min as previously described (Strick and Elferink, 2005
). Cells were acid-washed and then transferred to 37°C in DMEM/0.5% BSA containing 50 μg/ml unlabeled transferrin for the indicated time. Alternatively, cells were incubated for 10 min at 37°C after the 16°C internalization step to allow accumulation of labeled transferrin in the endocytic recycling compartment. Cells were then acid-washed and returned to 37°C to measure recycling by ELISA (Carter et al., 1993
For transfections, silencer Negative control 1 small interfering RNA (siRNA) and AAK1-specific (sense: 5′-GGUGUGCAAGAGAGAAAUCtt-3′; antisense: 5′-GAUUUCUCUCUUGCACACCtg-3′) siRNAs were obtained from Ambion (Austin, TX). tTA HeLa cells (1 × 105) were seeded into each well of a six-well dish containing glass coverslips. For transferrin recycling assays, cells were incubated overnight at 37°C in DMEM containing 10% fetal bovine calf serum. The next morning, cells were washed with PBS, and the medium was replaced with 800 μl of OptiMEM (Invitrogen). siRNA, 100 pmol, in 200 μl OptiMEM was then transfected into cells of each well using 3 μl oligofectamine (Invitrogen), according to the manufacturer's protocols. After a 24-h incubation, 2.0 ml DMEM containing 10% fetal bovine calf serum was added to each well. Seventy-two hours later, cells were assayed for transferrin recycling. In experiments where recycling was quantitated by ELISA, cells were incubated with control or AAK1-specific shRNA-encoded adenovirus for 48 h. After incubation, cells were processed for transferrin recycling as described above.
For rescue experiments, 3 × 105 tTA HeLa cells were plated onto a 35-mm dish containing OptiMEM and incubated 12 h. Cells were then transfected with Lipofectamine 2000 and one of two Steath siRNAs (Invitrogen) targeting the 5′ untranslated region (UTR) of AAK1: Steath siRNA 2: sense 5′-GAGCCGUCUCAAGUUUAAACUUACA-3′; antisense: 5′-UGUAAGUUUAAACUUGAGACGGCUC-3′ (siRNA 2) or Steath siRNA 3: sense 5′GCGCGAUUGACACGCAUAUUCCUAU-3′, antisense 5′-AUAGGAAUAUGCGUGUCAAUCGCGC-3′ (siRNA 3). Cells were incubated for 48 h, split into two 35-mm dishes containing growth medium (DMEM/5% fetal bovine serum [FBS]/400 μg/ml G418) supplemented with 10 ng/ml tetracycline, and then infected with tetracycline-regulatable adenovirus encoding either AAK1 CBD1 as a control or full-length AAK1L. Cells were incubated an additional 24 h before being assayed for transferrin recycling.