Goodpasture antigen-binding protein is a soluble exportable protein that interacts with type IV collagen. Identification of novel membrane-bound isoforms

Abstract

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Various studies have questioned these findings and proposed that GPBP serves as transporter of ceramide between the endoplasmic reticulum and the Golgi apparatus. Here we show that cells expressed at least two GPBP isoforms resulting from canonical (77-kDa) and noncanonical (91-kDa) mRNA translation initiation. The 77-kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment. The 91-kDa polypeptide and its derived 120-kDa polypeptide associated with cellular membranes and regulated the extracellular levels of the 77-kDa polypeptide. A short motif containing two phenylalanines in an acidic tract and the 26-residue Ser-rich region were required for efficient 77-kDa polypeptide secretion. Removal of the 26-residue Ser-rich region by alternative exon splicing rendered the protein cytosolic and sensitive to the reduction of sphingomyelin cellular levels. These and previous data implicate GPBPs in a multicompartmental program for protein secretion (i.e. type IV collagen) that includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization and 2) interorganelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane.

Figures

FIGURE 1.

COL4A3BP encodes for polypeptides of 77-, 91-, and 120-kDa. In A, FLAG-tagged GPBP or GPBPΔ26/CERT (10–20 ng) were analyzed by Western blot with the indicated antibodies. In B, cell extracts (50 μg) were analyzed as in A. In C, extracts (10 μg) from control cells (-) or cells expressing pc-n4′were analyzed as in A. In D, extracts (50 μg) from untransfected cells (-) or from cells transfected with the indicated siRNA-expressing plasmid were analyzed as in A. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control and siRNA specificity. The reactivity of mAb e26 with native or recombinant polypeptides was fully abolished when using GPBPpep1 (20 μm) as antibody blocking peptide (not shown). In this and the following figures, numbers and bars or arrows indicate the size in kDa and the positions of the molecular mass standards or the reactive polypeptides, respectively. The results shown in this and following figures are representative of at least two independent experiments.

FIGURE 2.

GPBP polypeptides of 91- and 120-kDa are the products of mRNA noncanonical translation initiation. In A, schematic representation of the cDNAs used to construct the indicated plasmids. In B, cell extracts (10 μg) (ex vivo) or individual transcription/translation mixtures (in vitro) expressing the indicated plasmid constructs were analyzed by Western blot using mAb e26 (ex vivo) or by fluorography (in vitro). Lysates from untransfected cells (ex vivo) or mixtures without plasmid (in vitro) were used as control. In C, indicated are the sequence of the N-terminal open reading frame of GPBP in one-letter code and the corresponding mRNA nucleotide sequence in capital letters. The gray and black letters indicate the 5′-UTR and ATR, respectively. Boxed are the codons and residues for canonical and noncanonical translation initiation. The peptide sequence targeted by Ab 24 is highlighted in gray. The negative numbers at the right denote the position of the codon or residue from canonical translation initiation site (AUG or Met, +1). In D, extracts (10 μg) from cells not expressing (Control) or expressing the indicated plasmid constructs without (-) or with a stop codon at the indicated positions were analyzed by Western blot using the indicated antibodies. In E, partially purified cell extracts (50 μg) were analyzed by Western blot using the indicated reactive species and a nonreactive F(ab)2 Ab 20 (Cont).

FIGURE 3.

The 91- and 120-kDa GPBP isoforms are insoluble membrane-bound polypeptides. In A, intact cells were incubated with αGPBP-Alexa Fluor 647 antibodies (αGPBP-AF647) in the presence of GPBPpep1 or equimolecular amount of a nonrelevant peptide (Contpep) and Rhodamine 123 for mitochondrial staining of living cells and analyzed by confocal microscopy. Scale bar, 21 μm. In B, the cells were detached and incubated with blocking solution in the absence (control) or presence of biotinylated αGPBP antibodies (αGPBP). The cell surface-bound antibody was detected with streptavidin-FITC and flow cytometry. As a control, parallel cultures were incubated with the same antibodies in the presence of GPBPpep1 (αGPBP + GPBPpep1) or equimolecular amount of a nonrelevant peptide (αGPBP + Contpep) and similarly analyzed. In C, similar amounts (10 μg) of the indicated cellular fractions were analyzed by Western blot using antibodies for the indicated proteins. We used as cellular compartment markers: pyruvate dehydrogenase (PDH) for mitochondria; cathepsin D for lysosome; prion protein (PrP) for microsome; and nuclear factor κB (p65) for nucleus and cytosol. For GPBP and GPBPΔ26/CERT detection, we used mAb e26 and mAb 14, respectively. Because we did not detect expression of 77-kDa GPBP in the cytosol, mAb 14 reactivity in this compartment can be attributed to GPBPΔ26/CERT.

FIGURE 4.

The 77-kDa GPBP isoform interacts with type IV collagen in cultured cells. In A, HEK 293 or HEK 293-FLAG-α3(IV) cells were cross-linked, lysed, and αFLAG extracted. Fifty micrograms of cell lysate (Input) or the corresponding FLAG-immunoprecipitated materials (IPαFLAG) were reversed cross-linked and analyzed by Coomassie Blue staining or Western blot with αGPBPr. The major specific polypeptides in FLAG-immunoprecipitates (arrows) were excised and identified by MALDI/TOF/TOF mass spectrometry. In B, HEK 293 (-) or HEK 293-FLAG-α3(IV) (+) cells were transfected with pcDNA3 (-) or with pc-n4′ (+), cross-linked, processed, and analyzed as in A by Western blot using the indicated antibodies.

FIGURE 5.

Export of 77-kDa GPBP to the extracellular compartment. In A, HeLa cells were transfected with the indicated plasmid constructs, and the indicated proteins were visualized by standard indirect immunofluorescence. DNA was stained with 4′,6′-diamino-2-phenylindole (DAPI) for nuclear visualization. Original magnification, ×400. In B and C, extracts (10 μg) from cells expressing the indicated plasmid constructs (lysates) or FLAG immunoprecipitates from the corresponding culture media (media IP) were analyzed by Western blot using the indicated antibodies.

FIGURE 6.

The 91-kDa GPBP regulates 77-kDa GPBP secretion in cultured cells. In A, extracts (10 μg) from two independent clones expressing (c8) or not expressing (c19) recombinant 91-kDa GPBP were analyzed by Western blot with mAb 14 antibodies, which react poorly with native 91-kDa counterpart (Fig. 1B). In B, the same clones were transfected with pc-FLAG-GPBP, and cell extracts (lysates) or FLAG immunoprecipitates from the corresponding culture media (media IP) were analyzed by Western blot using the indicated antibodies. Similar conclusions were obtained when assaying c14, an independent HEK 293 clone expressing levels of recombinant 91-kDa GPBP similar to c8 (not shown).

FIGURE 7.

GPBPΔ26/CERT but not GPBP is sensitive to sphingomyelinase cell treatment. In A, HeLa cells were transfected with the indicated plasmid constructs and treated (+) or not (-) with spingomyelinase, lysed, FLAG immunoprecipitated, and analyzed by Western blot with αFLAG antibodies (bSMase). Immunoprecipitates from untreated cells were incubated (+) or not (-) with phosphatase and similarly analyzed (λPPase). We have used a 8–12% gradient gel and extensive electrophoresis to separate phosphorylated and dephosphorylated versions of FLAG-tagged GPBPΔ26/CERT and estimated their relative abundance by Western blot and densitometry. In B, the same cells as in A were fixed by methanol/acetone, double-labeled with anti-FLAG-FITC antibody (green) and 4′,6′-diamino-2-phenylindole (blue), and analyzed by direct immunofluorescence. Original magnification, ×400.