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doi: 10.1242/10.1242/jcs.00184

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Trafficking of lysosomal cathepsin B—green fluorescent protein to the surface of thyroid epithelial cells involves the endosomal/lysosomal compartment

Martin Linke^1,*, Volker Herzog¹ and Klaudia Brix^1,2,

¹ Institut für Zellbiologie and Bonner Forum Biomedizin, Universität Bonn, Ulrich-Haberland-Str. 61a, D-53121 Bonn, Germany
² School of Engineering and Science, International University Bremen, PO Box 75 05 61, D-28725 Bremen, Germany
^* Present address: Department of Human Genetics, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA

View larger version (78K): [in a new window]   Fig. 1. Localization and possible function of cathepsin B in rat thyroid epithelial cells. A confocal fluorescence micrograph of a segment of a rat thyroid follicle (A), and a conventional fluorescence micrograph of formaldehyde-fixed (B) and Triton-X-100-permeabilized (C) FRT cells after immunolabeling with rabbit anti-rat cathepsin B antibodies. Cathepsin B was recognized within vesicles resembling endosomes or lysosomes (arrows) or in association with the plasma membrane (arrowheads) of rat thyroid epithelial cells in situ (A) and in vitro (B,C). In vitro degradation of Tg with plasma-membrane-associated proteases of FRT cells without (blue curve) or after inhibition of cysteine proteases by E64 (red curve) and identification of liberated thyroid hormones (eluting positions marked by green arrows) by reversed phase chromatography (D). Note that preincubation with E64 completely abolished liberation of T3 and T4 by proteases associated with plasma membrane preparations (D, cf. red with blue curve), indicating the contribution of cell-surface-associated cysteine proteases in Tg processing for thyroid hormone liberation. N, nuclei; stars, Golgi cisternae. Bars, 10 µm (A), 50 µm (B,C).

View larger version (6K): [in a new window]   Fig. 2. Schematic drawing of the chimeric protein CB-EGFP. The cathepsin B portion of the chimeric protein consists of the signal peptide (S, light grey), the propeptide (pro, dark grey) and the light (LC, blue) and heavy chains (HC, blue) of the protease, which are covalently linked to EGFP (green) by a spacer peptide (pink). Exchanges of two amino acids within the propeptide of cathepsin B from FRTL-5 cells are indicated in grey, the positions of two potential N-glycosylation sites in orange and the active site residues of the cathepsin B portion of the chimeric protein are shown in blue. The complete coding sequence of the vector pCathB-EGFP is available from GenBank under accession number AF490378.

View larger version (41K): [in a new window]   Fig. 3. Heterologous expression of CB-EGFP and its lysosomal localization in CHO cells. Single channel fluorescence (lower panels, middle and right), merged (A,B) and phase contrast micrographs (lower panels, left) of CHO cells transiently expressing CB-EGFP (green in A,B) were taken with a confocal LSM after immunolabeling with antibodies specific for heterologously expressed rat cathepsin B (A, red) and after pulse-chase loading of lysosomes with Lyso Tracker (B, red). Yellow signals are indicative of colocalization. N, nuclei. Bars, 20 µm.

View larger version (91K): [in a new window]   Fig. 4. Trafficking of CB-EGFP in FRT cells. Single channel fluorescence (lower panels, middle and right, and inset in D), merged (A-D) and phase contrast micrographs (lower panels, left) of FRT cells transiently expressing CB-EGFP were taken with a confocal LSM after immunolabeling with antibodies against the ER-resident protein PDI (A, red), the Golgi mannosidase II (B, red), the endogenous lysosomal cysteine protease cathepsin B (D, red) and after pulse-chase loading of lysosomes with Lyso Tracker (C, red). For reference, the inset in D shows non-transfected FRT cells after immunolabeling of cathepsin B. Yellow signals are indicative of colocalization, arrowheads point to ER-cisternae, stars mark the positions of the Golgi and arrows indicate lysosomes. N, nuclei. Bars, 40 µm.

View larger version (92K): [in a new window]   Fig. 5. Trafficking of CB-EGFP in FRTL-5 cells. Single channel fluorescence (lower panels, middle and right, and inset in D), merged (A-D) and phase contrast micrographs (lower panels, left) of FRTL-5 cells transiently expressing CB-EGFP were taken with a confocal LSM after immunolabeling with antibodies against the ER-resident protein PDI (A, red), the Golgi mannosidase II (B, red), the endogenous lysosomal cysteine protease cathepsin B (D, red) and after pulse-chase loading of lysosomes with Lyso Tracker (C, red). For reference, the inset in D shows non-transfected FRTL-5 cells after immunolabeling of cathepsin B. Yellow signals are indicative of colocalization. Arrowheads point to ER-cisternae; stars mark the positions of the Golgi and arrows indicate lysosomes. N, nuclei. Bars, 40 µm.

View larger version (61K): [in a new window]   Fig. 6. Transport of CB-EGFP in living FRT cells. Single channel fluorescence (A,B,D'',D'''), merged (C,D) and phase contrast micrographs (D') of FRT cells transiently expressing CB-EGFP taken in the conventional (A) or the confocal mode (B-D''') at 37°C (A,C), 20°C (B) or after fixation and immunolabeling with antibodies against rat lgp96 (D). During steady state, CB-EGFP was detectable within the lumen of the ER, the Golgi apparatus and within vesicles (A). Note, the accumulation of CB-EGFP within the TGN after incubation of the cells at 20°C (B), its subsequent transport to vesicles after shifting to the transport-permissive temperature of 37°C (C) and its colocalization with the lysosomal marker lgp96 (D). Micrographs of different focal planes are merged in C, and colored in blue, green and red as indicated. Yellow signals in D are indicative of colocalization of green fluorescent CB-EGFP (D''') with lgp96 (D''). Arrowheads point to ER-cisternae, stars mark the positions of the Golgi and the TGN and arrows indicate lysosomes. N, nuclei. Bars, 50 µm (A,C), 20 µm (B,D,D').

View larger version (25K): [in a new window]   Fig. 7. Lysosomal CB-EGFP and its secretion from transfected cells. Lysates of lysosomal fractions of non-transfected (lanes 3, 5, 7, 10, 12, 14) or CB-EGFP-expressing CHO, FRT or FRTL-5 cells after selection with G418 (lanes 4, 6, 8, 11, 13, 15) were normalized to contain equal amounts of protein and separated on 12.5% SDS gels. After blotting, proteins were immunolabeled with antibodies against rat cathepsin B (A) or GFP (B). Recombinant human procathepsin B (lane 1), bovine spleen cathepsin B (lane 2) and EGFP (lane 9) were used as standards. C shows an autoradiograph of SDS-PAGE-separated anti-GFP immunoprecipitates from culture media collected after the indicated time intervals from radiolabeled non-transfected (lane 18) or CB-EGFP-expressing FRT cells (lanes 16 and 17). Molecular mass markers are given in the left margin. The positions of the intact chimeric protein (CB-EGFP) and its degradation fragment (F1) as well as those of procathepsin B (pro), single chain (SC) and heavy chain of two-chain cathepsin B (HC) are indicated in the right margins. The proteolytic activity of cathepsin B within conditioned media of transfected (+) or non-transfected CHO, FRT or FRTL-5 cells was determined at pH 6.0 by using a colorimetric assay (D). Cathepsin B activities in D are given as mean±standard deviation; levels of significance are indicated as ** for P<0.01, n=3.

View larger version (23K): [in a new window]   Fig. 8. Stimulated secretion of lysosomally matured CB-EGFP from FRTL-5 cells. Lysates of non-transfected (A, lane 1) or CB-EGFP-expressing FRTL-5 cells (A, lane 2) were normalized to provide equal amounts of protein and separated on 12.5% SDS gels for subsequent blotting and immunolabeling with antibodies against the propeptide of rat cathepsin B. Autoradiography of 12.5% SDS gels of secretion media from CB-EGFP-expressing, G418 selected FRTL-5 cells after 1 hour of pulse radiolabeling (B, lanes 3 and 4) and chasing for the indicated time intervals in media without TSH (B, 5H, lanes 5-10) or with 50 µU/ml TSH (B, 5H + TSH, lanes 11-16). Immunoprecipitation was with antibodies against rat cathepsin B (CB, odd numbered lanes in B) or against the propeptide of rat cathepsin B (PP, even numbered lanes in B). Molecular mass markers are given in the margins. The positions of the proform (proCB-EGFP) and the mature chimeric protein (CB-EGFP), as well as of procathepsin B (pro) and single chain cathepsin B (SC) are indicated in the margin between A and B. In C, the proteolytic activity of CB-EGFP secreted from continuously TSH-stimulated FRTL-5 cells after immunoprecipitation with anti-GFP antibodies is given as mean±standard deviation. Note that antibodies against the propeptide of cathepsin B failed to immunoprecipitate CB-EGFP from the secretion media (B) and that secreted CB-EGFP was proteolytically active (C).

View larger version (23K): [in a new window]   Fig. 9. Transport pathways of lysosomal enzymes in rat thyroid epithelial cells. Schematic drawing summarizing the results on lysosomal enzyme trafficking in FRT or FRTL-5 cells. The occurrence of mature cathepsin B at the surface of thyroid epithelial cells might be explained by extracellular processing of secreted procathepsin B (light green) to the mature enzyme (dark green, left portion, crossed arrows). This report shows that intralysosomal processing was the prerequisite for the retrograde transport of mature cathepsin B from lysosomes to the apical plasma membrane and its subsequent secretion into the extracellular space (dark green, right portion, bold arrow). Extracellularly occurring CB-EGFP was resistant to proteolytic degradation for long time intervals of up to two days, suggesting the stability of secreted lysosomal enzymes, thus explaining their function in the extracellular proteolysis of Tg at the surface of thyroid epithelial cells.