Cytochemical demonstration of expression and distribution of non-glycosylated human lysosomal cathepsin S in HEK 293 cells.
The lysosomal cysteine protease cathepsin S is synthesized as inactive precursor at the rough endoplasmic reticulum (ER), further processed in the Golgi compartment and finally targeted to the lysosomes where it becomes activated by the proteolytic cleavage of the inhibitory propeptide. Biochemical studies with a non-glycosylated mutant of procathepsin S (plasma membrane binding at 2 degrees C, reuptake of secreted enzyme at 37 degrees C) led to the suggestion of an additional sorting motif in procathepsin S besides the classical Man-6-P recognition signal. In order to further confirm this suggestion on a morphological basis we performed a series of laser scanning confocal microscopy (CLSM) and electron microscopical analyses with HEK 293 cells expressing the mutant non-glycosylated procathepsin S. Immunolocalization with CLSM documented clearly a fine granular fluorescence in the paranuclear region of mutant expressing cells. Electron microscopy demonstrated the presence of cathepsin S immunoreactive deposits within cytosolic vacuoles (lysosomes), at the plasma membrane and in ER buds. These buds were also visible in the cytosol as well as in form of concentrated patches at the plasma membrane indicating the direct transport of (pro)cathepsin S from the ER to the cell surface.
Nissler
Karl
K
Institute of Biochemistry, Friedrich-Schiller-University, Nonnenplan 2, 07743 Jena, Germany. knis@mti-n.uni-jena.de
Oehring
Hartmut
H
Krieg
Reimar
R
Pierskalla
Alexandra
A
Weber
Ekkehard
E
Wiederanders
Bernd
B
Halbhuber
Karl-Jürgen
KJ
D000135:Acid Phosphatase / Q000378:metabolism; D001483:Base Sequence; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007668:Kidney; D008247:Lysosomes / Q000201:enzymology* / Q000648:ultrastructure; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D000135:Acid Phosphatase; D002403:Cathepsins; C028217:cathepsin S