Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells.
Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating "pro(C)"-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.
Källquist
Linda
L
Department of Hematology, BMC C14, Lund University, SE-221 84 Lund, Sweden.
Rosén
Hanna
H
Nordenfelt
Pontus
P
Calafat
Jero
J
Janssen
Hans
H
Persson
Ann-Maj
AM
Hansson
Markus
M
Olsson
Inge
I
D033963:Adaptor Protein Complex 3 / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000648:ultrastructure; D004705:Endocytosis; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology; D019272:Leukocyte Elastase / Q000737:chemistry / Q000378:metabolism*; D009000:Monocytes / Q000201:enzymology* / Q000473:pathology; D053146:Myeloblastin / Q000737:chemistry / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000473:pathology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D051381:Rats; D022021:Tacrolimus Binding Proteins / Q000378:metabolism; D060149:Tetraspanin 30; D014443:Tyrosine / Q000378:metabolism
D033963:Adaptor Protein Complex 3; D015703:Antigens, CD; C554586:CD63 protein, human; C554588:Cd63 protein, rat; D004792:Enzyme Precursors; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30; D014443:Tyrosine; D019272:Leukocyte Elastase; D053146:Myeloblastin; D022021:Tacrolimus Binding Proteins