Identification of the BAP31-associated protein PTPLB in the novel split-ubiquitin yeast two-hybrid screen. (A) Diagrammatic representation of the split-ubiquitin two-hybrid system. Polypeptides that interact with the bait protein bring together the C-terminal fragment (Cub) and a modified N-terminal fragment (NubG, isoleucine 13 replaced by glycine) of ubiquitin, allowing ubiquitin-specific proteases (UBPs) to liberate PLV (protein A, LexA, and VP16) from Cub, which then is free to enter the nucleus (NU) and activate transcription of the lacZ and HIS3 reporter genes. (B) Growth of yeast L40 cells expressing BAP31-Cub-PLV, NubG, or NubG-PTPLB fusion proteins alone or together on various selective agar plates. BAP31-Cub-PLV in a leucine selection vector was integrated at the Cup1 genetic site in L40 yeast cells (pRSB-L40). NubG and NubG-PTPLB were constructed into a 2μm plasmid with tryptophan selection and transfected into L40 or pRSB-L40 yeast cells. The yeast strains containing various constructs were inoculated on agar plates with different selections. All the agar plates contained 0.2 mM Cu²⁺ with or without 3-aminotriazole (AT). (C) Western blot analysis of yeast L40 cell lines expressing BAP31-Cub-PLV fusion protein with or without coexpression of the NubG or NubG-PTPLB vector in the presence or absence of added Cu²⁺. Cells were grown to the logarithmic phase in relative selection medium. Proteins were extracted and analyzed by Western blotting with rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugate. L, lysine; T, tryptophan; H, histidine; full, no selection.

Molecular cloning of human PTPLB cDNAs. (A) Representative clones covering the full-length cDNA of the PTPLB gene are shown. An asterisk indicates the translation stop codon. The open box represents the open reading frame identified in the two-hybrid screen; the 5′ and 3′ untranslated sequences were determined by 5′- and 3′-RACE and expressed sequence tag human GenBank overlaps. (B) PTPLB is composed of six exons. The locations of the six exons are indicated. (C) Proposed topology of PTPLB in the ER membrane, as predicted by hydropathy, the “positive inside” rule, and the known cytosolic disposition of C-terminal ER retention signals (see reference 34). (D) Nucleotide and deduced amino acid sequences of human PTPLB. The amino acid sequences are shown in the single-letter code. The translation stop codon is indicated by an asterisk. The PTPase homology motifs, HCXXGXXRS and an aspartic acid (D), are indicated by bold letters, and the proline that replaces the catalytic arginine is bold and underlined. The C-terminal ER retention motif, KKFE, is in bold. Four transmembrane segments are predicted by hydropathy plot analysis, performed with the TMHMM software, and the sequences are indicated by the heavy underlines.

Northern blot analysis of PTPLB gene expression. The membranes contained 2 μg each of high-quality polyadenylated mRNA isolated from the indicated human tissues and organs. Hybridization was done with the ³²P-labeled DNA fragment of the PTPLB open reading frame (upper panels) and a β-actin probe as a control (lower panels). PBL, peripheral blood leukocytes.

PTPLB turns over via the ER-associated degradation pathway. (A) Cos-1 cells were transfected with vector alone or vector encoding PTPLB-Myc-KKFE or PTPLB-HA-KKFE, and after 18 h, MG132 (100 μM) was added to the medium for 4 h and the cells were analyzed by immunofluorescence microscopy. (B) Cos-7 cells were transfected with PTPLB-Myc-KKFE, and 18 h later, lactacystin (5 μM) was added for the indicated times. Cells were analyzed by immunofluorescence microscopy with 20× (top panel) or 100× (lower panel) lenses. (C) Cos-1 cells were transfected with vector encoding wild-type PTPLB-HA (Wt), PTPLB-HA-0K, and N-Myc-PTPLB-HA, and after 18 h, MG132 (100 μM) was added to the medium for 4 h and the cells were analyzed by immunofluorescence microscopy with a ×20 or a ×100 lens. The scale bar in the pictures taken at 20× magnification represents 125 μm (top panels), whereas in the pictures taken at 100× magnification it represents 25 μm (lower panels). (D) Cos-1 cells were transfected with the vector, PTPLB-HA, or long N-Myc-PTPLB-HA. After 18 h, cell extracts containing equivalent amounts of protein were assessed by immunoblotting of anti-HA immunoprecipitates (IP) with anti-HA antibodies. (E) Stability of PTPLB and its mutants in vivo. The half-life of wild-type PTPLB, lysine-free PTPLB-0K (lysine residues were replaced with arginines), and N-Myc-PTPLB in Cos-1 cells was measured in a pulse-chase experiment, followed by immunoprecipitation of the labeled proteins as described in Materials and Methods. The proteasome inhibitor MG132 (100 μM) was added at 30 min after the addition of [³⁵S]methionine-[³⁵S]cysteine and was present throughout the 2-h chase period (lanes 4, 8, and 12). (F) Quantitative (PhosphorImager) analysis of the data depicted in panel E after the indicated chase periods in the absence and presence of the proteasome inhibitor. Quantities are relative to the amount of the indicated ³⁵S-labeled PTPLB proteins at time zero.

PTPLB associates and colocalizes with BAP31 in the ER. (A) Cos-1 cells grown on coverslips were transfected with PTPLB-HA for 18 h, followed by the addition of MG132 to the medium for 4 h, and the cells were double stained with anti-HA antibody (green) and either anti-BAP31 (top panel), anti-calnexin (ER marker, middle panel), or anti-TOM20 (mitochondrion marker, bottom panel) (red), and images were visualized in the red, green, and yellow (overlay) channels. (B) Association of PTPLB with BAP31 in Cos-1 cells. Cos-1 cells were transfected with cDNAs coding for N-Myc-PTPLB-HA and/or BAP31-Flag as indicated. Anti-HA immunoprecipitates (IP) were recovered and resolved by SDS-PAGE, and immunoblots were probed with anti-Flag (top panel) or anti-HA (middle panel). Equivalent samples from cell lysates were immunoblotted with anti-Flag (bottom panel). (C) Small amounts of PTPLB associate with BAP31 in Cos-1 cells. Cos-1 cells were transfected with cDNAs coding for N-Myc-PTPLB-HA with or without BAP31-Flag as indicated. Anti-Flag immunoprecipitates (IP) were recovered and resolved by SDS-PAGE, and immunoblots were probed with anti-HA (lanes 1 and 2). Subsequently, the remaining PTPLB molecules were collected from the BAP31-depleted supernatants with an anti-HA antibody, and the immunoblots were probed with anti-HA (lanes 3 and 4).

Immune complex phosphatase assays. Cos-1 cells were transfected for 18 h with cDNAs coding for N-Myc-PTP1B, N-Myc-PTP1B-D24A, N-Myc-PTPLB, and N-Myc-PTPLB-P101R. The protein expression levels were monitored by immunoblotting of the anti-Myc immunoprecipitates with anti-Myc antibodies, and the abundances of the proteins were adjusted to the same levels. The PTPase activity assay was performed as described in Materials and Methods. The absorbance at 410 nM represents the quantities of the dephosphorylated product of pNPP.

BAP31 maintains PTPLB expression levels. (A) Equivalent samples of cell lysates from Bap31-null and Bap31-null/BAP31 cell lines were analyzed by immunoblotting with anti-BAP31 and anti-γ-actin antibodies, as indicated. (B) The two cell lines were transfected with vectors expressing GFP or PTPLB-GFP, and GFP was monitored by immunofluorescence microscopy. (C) The Bap31-null and Bap31-null/BAP31 cell lines were transfected with vector pcDNA3.1 or a vector expressing GFP or PTPLB-GFP. After 36 h, the cells were fixed in 1% paraformaldehyde in phosphate-buffered saline for 30 min and analyzed by flow cytometry. The data were gated (R1) to exclude emissions obtained in the vector control cells.