Secretion of Polypeptide Crystals from Tetrahymena thermophila Secretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin, Cth4p

Expression and localization of CFP-tagged Cth4p. (A) Western blot, probed with anti-GFP MAb that cross-reacts with CFP, of cells expressing CFP-tagged Cth4p (cth4-1p). cth4-1p expression was induced for 30, 60, and 120 min with 1 μg/ml of CdCl₂. At these times, cell lysates were prepared by trichloroacetic acid (TCA) precipitation and then solubilized in SDS-PAGE sample buffer. Proteins fractions were separated by 4 to 20% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). Molecular mass standards are shown on the left. The specific bands recognized by the antibody, indicated by arrowheads, are of the size expected for full-length Cth4p-CFP (94 kDa) and monomeric CFP. At 30 and 60 min of induction, only full-length Cth4p-CFP was detected, whereas monomeric CFP was also detected after 120 min. Asterisks indicate nonspecific cross-reactive species. (B) Using the same cultures as for panel A, cells at 30, 60, and 120 min were fixed and immunolabeled with mouse MAb 5E9 to localize mucocyst protein Grl3p and rabbit anti-GFP to localize the Cth4p-CFP fusion. The fluorescent puncta at the cell surface, which appear as elongated vesicles in cross section, correspond to the array of docked mucocysts. Scale bars = 10 μm.

Localization of endogenous Cth4p-GFP. (A) Expression of cth4-2: GFP-tagged Cth4p from the endogenous CTH4 locus, under the control of its native promoter. cth4-2p was immunoprecipitated from detergent lysates using polyclonal rabbit anti-GFP antiserum, and immunoprecipitates were Western blotted with monoclonal anti-GFP Ab. One immunoreactive band is of the size expected for the Cth4p-GFP fusion, while a second is the size expected for monomeric GFP. (B) Live immobilized cells expressing cth4-2p were imaged to capture cell surface and cross sections and show the expected array of docked mucocysts as well as intracellular puncta. (C) Immunostaining of fixed cells to simultaneously localize mucocyst protein Grl3p and cth4-2p. There is extensive colocalization in docked mucocysts, while some puncta deeper in the cytoplasm are positive for cth4-2p but not Grl3p. (D) Cells were incubated for 5 min with 200 nM Lysotracker, and live images were captured within 30 min. Optical sections shown are cell cross sections. (E) Cells were incubated for 5 min with 5 μM FM4-64, an endocytic tracer and then pelleted and resuspended in tracer-free medium. The times shown represent minutes after resuspension. Scale bars = 10 μm.

Monomeric GFP, but not Cth4p-GFP, is released upon mucocyst exocytosis. Fifty-milliliter cultures of wild-type or cth4p-GFP expressing cells (from the endogenous locus) were first grown overnight to ∼3 × 10⁵/ml at 30°C in SPP and then incubated for an additional 24 h at 22°C with gentle shaking. Two 15-ml samples were removed from each culture and pelleted and resuspended in 1.5 ml of medium, and one half were treated with dibucaine for 20 s. The cells were then diluted to 15 ml and centrifuged to generate cell pellet, flocculent, and supernatant fractions as in Fig. 5. After aspiration of the superatants, tubes were sliced with a razor below the flocculent-cell interface, and the flocculent was discarded. Cells were resuspended in 10 mM Tris (pH 7.4), repelleted, and then lysed with 10%TCA. Lysates (5 × 10⁴ cell equivalents) were resolved by 4 to 20% SDS-PAGE, transferred to PVDF, and Western blotted with anti-GFP (A) or anti-Grl1p (B) antibody. (Left blot) No release of cth4p-GFP from cells is seen, since the band intensities are equivalent in untreated and dibucaine-treated cells. In contast, a substantial fraction of free GFP is released by dibucaine stimulation. (Right blot) Grl1p is released from wild-type or cth4p-GFP cells during dibucaine stimulation.

CTH4 is required for normal mucocyst discharge. (A) Schematic of CTH4 gene knockout construct. Replacement of the CTH4 gene by the Neo4 drug resistance cassette, to generate Δcth4, was targeted by homologous recombination. (B) Verification of gene knockout by RT-PCR. RNA was extracted from wild-type (WT) and Δcth4 cells, subjected to coupled reverse transcription, and PCR amplified using primers listed in Table S1 in the supplemental material. As shown in this 1% ethidium bromide-stained agarose gel, Δcth4 cells lacked any detectible products corresponding to the targeted gene. SOR3 provides a positive control. (C) Illustration of semiquantitative assay for mucocyst discharge. Cells were stimulated with dibucaine for 20 s, which triggers synchronous exocytosis of mucocyst contents. Subsequent centrifugation results in a dense pellet of cells, with an overlying flocculent comprised of expanded cores of exocytosed mucocysts. (D) Stimulated WT cell cultures generate the expected two-layered pellet (left). For clarity, the flocculent layers are delineated with a dashed line at the lower border and an unbroken line at the upper border. Stimulated Δcth4 cultures produce a greatly reduced amount of flocculent (right). Although identical numbers of cells were used in all samples, the poststimulation wild-type cell pellet is smaller because some cells have remained trapped in the sticky flocculent layer.

CTH4 is not required for formation and accumulation of docked mucocysts. (A) (Top) docked mucocysts in fixed wild-type cells, immunolabeled using MAb 4D11, which recognizes Grt1p (left two images), and MAb 5E9, which recognizes Grl3p (right two images). Shown are optical surface and cross sections. (Bottom) parallel immunostaining of Δcth4 cells shows patterns indistinguishable from the wild type. Scale bars = 10 μm. (B) Electron micrographs of mucocysts (labeled with asterisks) in wild-type and Δcth4 cells. The mucocyst cores in both wild-type and Δcth4 cells are organized as visible lattices. Scale bars = 0.2 μM.

CTH4 is required for efficient cargo release upon exocytosis. (A) Illustration of qualitative assay for mucocyst discharge. Cells are stimulated by addition of alcian blue, which triggers mucocyst exocytosis and binds to released mucocyst proteins, resulting in two distinct pools of cells. Cells in pool 1 are individually trapped by translucent capsules formed by released mucocyst contents, while cells in pool 2 are not entrapped because they have already escaped from their capsules. (B and C) Cells, before and after stimulation with alcian blue, were fixed, detergent permeabilized, immunolabeled with MAb against Grl3p, and then analyzed by confocal microscopy or flow cytometry. (B) Wild-type and Δcth4 cells prior to stimulation display identical docked mucocysts, visible as discrete puncta (top two rows). After stimulation, wild-type cells are surrounded by translucent capsules of released mucocyst contents, distinct from the punctate mucocyst staining prior to stimulation, that stain brightly for Grl3p (pool 1, 3rd row) or are largely devoid of Grl3p staining (pool 2, 4th row). Stimulated Δcth4 cells do not form capsules and still display abundant docked mucocysts (bottom row), though different in appearance from prestimulation samples. Scale bars = 10 μm. Insets in right-hand panels: rows 1 and 2, elongated docked mucocysts in WT and Δcth4 cells prior to stimulation; row 5, irregular mucocyst profiles in Δcth4 cells following stimulation. (C) Flow cytometry of wild-type and Δcth4 cells, before and after stimulation. (Left graph) prior to stimulation, WT and Δcth4 cells show similar labeling with anti-Grl3p Ab. (Right graph) after stimulation, WT cells show two distinct populations, corresponding to pools 1 and 2. Poststimulation Δcth4 cells instead show a single population, whose staining intensity suggests that roughly half of Grl3p was released by exocytosis. For flow cytometry, 10⁴ cells/sample were analyzed. MFI, mean fluorescence intensity. (D) Electron micrographs of docked mucocysts (*) in Δcth4 cells, fixed before and after alcian blue stimulation. Scale bars = 0.2 μm.

Secreted mucocyst contents from Δcth4 cells show increased solubility. (A) Illustration of samples following dibucaine stimulation of WT and Δcth4 cultures. Two-milliliter quantities of supernatants were withdrawn and precipitated with TCA, while 20-μl quantities of the flocculent layers were directly dissolved in SDS-PAGE buffer. (B and C) Western blots, using antibodies against Grl1p. The arrow indicates proGrl1p, and the arrowhead indicates processed Grl1p. (B) Supernatant fractions; (C) flocculent fractions. Δcth4 cells release both proGrl1p and mature Grl1p, with much of the protein found in the nonpelleting supernatant fraction. The comparison between flocculent sample lanes in WT versus Δcth4 cultures does not indicate similar amounts of release, since the volume of flocculent released by WT cultures is much greater than by Δcth4 cultures.

CTH4 is required for full processing of Grl proteins. Cell lysates (5 × 10³ cell equivalents in panel A and 10⁴ cell equivalents in panel B) were resolved by SDS-PAGE (10% [A] and 4 to 20% [B]), transferred to PVDF, and Western blotted with antibodies against Grl proteins. The unprocessed (proGrl) and processed (Grl) bands are labeled. (A) Blotting with anti-Grl1p antibody. The predominant band in WT lysates (lane 1) is mature processed Grl1p. The corresponding band in Δcth4 lysates is shifted slightly in mobility, and the lysate also shows a high level of unprocessed precursor. (B) Same as panel A, but blotting with anti-Grl3p antibody. (C) Gel (4 to 20%) stained with Coomassie brilliant blue, containing flocculent samples from dibucaine-stimulated WT and Δcth4 cultures. Molecular mass standards are shown on the left. The gel regions excised for mass spectrometry are shown. (D) The processed Grl products retain N-terminal extensions in Δcth4 cells. Each of the proGrl genes shown begins with an N-terminal signal sequence (gray rectangle) and has either one (for Grl1, -3, -5, and -7) or two (for Grl4) sites (blue circles) that were previously established as N termini of processed Grl products isolated from extruded wild-type mucocysts (20). The red rectangles represent the positions of peptides present in Δcth4 samples but not the wild-type samples. In each case, the peptide constitutes an amino-terminal extension to the N terminus in proteins isolated from wild-type cells. The amino acids within the Δcth4-specific peptides are shown below the corresponding rectangles in red, while the adjacent amino acids in blue are those at the beginning of the mature polypeptide in wild-type cells.

CTH4 activity requires conserved active site residues. (A) Schematic of wild-type and mutant Cth4p, both with C-terminal GFP tags, showing locations of mutations (Cys³⁵²→Ala and His⁵⁰⁵→Ala) within the conserved catalytic triad. (B) Expression of GFP-tagged and mutant Cys³⁵²→Ala and His⁵⁰⁵→Ala constructs (cth4-2 and cth4-3, respectively). Constructs were expressed at the CTH4 locus, via gene replacement. Fusion proteins were immunoprecipitated from detergent lysates using rabbit anti-GFP antiserum, transferred to PVDF, and probed with MAb anti-GFP. The transformed cell lines show immunoreactive bands of the size expected for the Cth4p-GFP fusions, as well as a band likely to correspond to monomeric GFP. (C and D) proGrl processing in wild-type and mutant cell lines. Cell lysates (5 × 10³ cell equivalents in panel C and 10⁴ cell equivalents in panel D) were separated by SDS-PAGE and probed with anti-Grl1p antibody. Wild-type and cth4-2 cells accumulate mature Grl1p. In contrast, Δcth4 cells and cth4-3 cells accumulate proGrl1p and a species slightly larger than mature wild-type Grl1p. (D) Same as panel C, but with blotting with anti-Grl3p antibody. (E) Dibucaine stimulation of cell lines in panels C and D. Wild-type cells and cells expressing cth4-2p generate large flocculent layers (between the solid and broken lines), while Δcth4 and cells expressing cth4-3p show a smaller amount of flocculent. (F) Cells expressing cth4-3p were fixed, permeabilized, and immunostained for GFP and for Grl3p. Localization of the mutated cth4-3p is similar to that of the wild-type enzyme, in both cytoplasmic puncta and docked mucocysts. Scale bars = 10 μm.

Model for proteolytic processing of mucocyst proGrl proteins in Tetrahymena. We propose that processing of proGrl proteins during mucocyst maturation initiates with endoproteolytic cleavage by Cth3p at a site upstream of the mature N termini. The products are competent to form highly ordered complexes that assemble to create the elongated mucocyst core. Cth4p subsequently acts to trim the amino termini. This step is proposed to be essential for reinforcing the lattice in a way that promotes stability during expansion. Since removal of the peptides results in stabilization during expansion, we have termed them “destabilizing peptides” (DP). The relative timing of DP removal versus assembly is unknown. Cores that have not undergone DP trimming are defective during the directional expansion required for efficient core extrusion during exocytosis.