(a) One-letter amino acid sequence of PAT1. The region of PAT1 predicted to form a coiled-coil is shown in the shaded box, and the four imperfect KLC-like tandem repeats are in the open boxes. Putative protein kinase C phosphorylation sites are denoted by •. (b) Alignment of the KLC-like repeats. The four 42-amino acid, imperfect KLC-like tandem repeats in PAT1 aligned with KLC repeats from various organisms. The numbers indicate the position of amino acids in PAT1 sequence. C-KLC , Caenorhabditis elegans (accession no. P46822); s-KLC, squid (accession no. P46825); D-KLC, Drosophila (accession no. P46824); ch-KLC, chicken (accession no. 1208772); h-KLC, human (accession no. Q07866). An appendix with information on sequence analysis, alignments, etc., is published as supplemental data on the PNAS web site (www.pnas.org).

Interaction of PAT1 prey with APP-BaSS and various baits. (a) β-Galactosidase color assay: Yeast diploid cells (L40A5xEGY48) containing prey vector (pJG4–5) expressing PAT1 and bait vectors (p1979A) containing either the wild-type APP-BaSS (Tyr) or the mutant APP-BaSS (Ala) were grown on a galactose + histidine plate in the presence of -bromo-4-chloro-3-indolyl β-d-galactoside. These and other data are obtained from a pooled population of at least 5 colonies. (b) Growth assay: Yeast diploid cells obtained as above were grown on galactose plates lacking histidine with or without 20 μM 3-amino-1, 2,4-triazole (3-AT). (c) Quantitative β-galactosidase assay: Yeast diploid cells obtained as above were grown in galactose-containing liquid culture for 12–18 h, and the β-galactosidase activity was determined and normalized for cell density. Data represent average results from five individual colonies. Error bars indicate the standard deviation. Yeast cells (L40A5) transformed with the wild-type APP-BaSS bait vector alone were used as a negative control. (d) YSV90 yeast cells were cotransformed with PAT1 prey vector (in pJG4–5) and indicated bait vectors (in p1979A), and the transformants were grown on galactose-lacking leucine for 2–3 days. The names of proteins and the signaling activity of the sequence (BaSS or endocytic) are given on the left. Signal sequences fused to the DNA-binding domain of LexA are given on the right, and the residues crucial for signaling activity are underlined.

PAT1 exists as an oligomeric complex, associates with the membrane and cofractionates with APP. (a) Gel filtration: Cytosol prepared from COS-1 cells transiently transfected with PAT1 in pFLAG-CMV-2 vector was separated on a Superose 12 gel filtration column. The fractions were Western blotted and probed with anti-FLAG M5 antibody and detected by alkaline phosphatase reaction. The elution positions of protein standards (in kDa) are indicated. (b) Chemical cross-linking: COS-1 cells mock-transfected (lane 1) or transiently transfected with FLAG-PAT1 in (lanes 2 and 3) were treated without (lane 2) or with (lane 3) 10 mM dimethyl pimelimidate (DMP, Pierce) at 4°C for 60 min. After the reaction was quenched, the cells were directly lysed in SDS/PAGE sample buffer and analyzed by SDS/PAGE and Western blotting as above. ∗ indicates the cross-linked band of 160–180 kDa detected by M5 antibody. The additional bands may be higher oligomers. (c) Subcellular fractionation: FLAG-PAT1 transfected COS-1 cells were homogenized and centrifuged at 150,000 × g (TLA 120.2, Beckman), and the supernatant (lane 1) and the pellet (lane 2) fractions were analyzed by SDS/PAGE and Western blotting. Proportion of PAT1 in pellet fraction varied between 50% and 75%. (d) Cofractionation of PAT1 and APP. A postnuclear supernatant from COS-1 cells transfected with FLAG-PAT1 and APP was fractionated by sucrose gradient centrifugation, and the fractions were analyzed by SDS/PAGE and Western blotting and probed with anti-APP (22C11; upper panel) or anti-FLAG antibody (lower panel). Note that a portion of PAT1 cofractionates with APP (fraction 8, indicated by ∗). The quantitation of these data is presented on the PNAS web site (www.pnas.org) as Fig. 3e.

PAT1 is a cytoplasmic protein enriched in the perinuclear region. (a–c) Paraformaldehyde-fixed HeLa cells were labeled with mAb26 (a and b) and visualized by using the TSA system with HRP-conjugated secondary antibody and Cy3-tyramide. Cells in b are were also double-labeled with anti-tubulin antibody and visualized with fluorescein isothiocyanate-labeled secondary antibody (c). Note that PAT1 gives a punctate cytoplasmic staining, and in some cells the staining is clearly enriched in the perinuclear region (arrows in a and b) which colocalilizes with the microtubule organization center (MTOC) as detected by heavily concentrated microtubules (arrow in c). (Bar = 10 μm.) (d–f) Acetone-fixed HeLa cells were double-labeled with mAb26 (d) and anti-APP antibody (e) and visualized without TSA technique with rhodamin and fluorescein isothiocyanate-labeled secondary antibodies, respectively. The filamentous labeling of PAT1 is more obvious in these cells (arrows), which overlap with APP (arrowhead). The overlap of APP and PAT1 distribution is clearly seen as the orange signal in the merged image (f). The data showing the antibody specificity are presented on the PNAS web site (www.pnas.org) as Fig. 4 g and h.

PAT1 interacts with microtubules and affects APP trafficking. (a) Microtubule binding assay: Cytosol from transfected COS-1 cells was incubated with (lanes 1 and 2) or without (lane 3 and 4) taxol-stabilized microtubules in the presence of 1 mM AMP-PNP (lanes 1 and 3) or 10 mM Mg-ATP (lanes 2 and 4). Microtubules were pelleted through a 25% sucrose cushion, and the pellet was analyzed by SDS/PAGE and Western blotting as described in Fig. 3a and probed with anti-KHC antibody, anti-KLC antibody, and with M5 antibody. (b) COS-1 cells were cotransfected with HRP/βA4/TM/CD plasmid and control-plasmid or sense-plasmid (S) to overexpress and antisense-plasmid (A) to down-regulate cellular PAT1 levels. After 36–48 h, HRP activity released in the medium was assayed as described (12). HRP activity is expressed as percent of control and the results are from duplicate measurements of four independent experiments. Error bars show standard deviation.