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PMID: 7642697 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells.

The Journal of cell biology ·Vol. 130 ·No. 4 ·1995-08-00 ·Pages 781-96

Brown WJ, DeWald DB, Emr SD, Plutner H, Balch WE

Abstract

Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene, in the targeting of newly synthesized proteins to the vacuole, an organelle functionally equivalent to mammalian lysosomes (Schu, P. V., K. Takegawa, M. J. Fry, J. H. Stack, M. D. Waterfield, and S. D. Emr. 1993. Science [Wash. DC]. 260:88-91). The activity of Vps34p kinase is significantly reduced by the PI 3-kinase inhibitors wortmannin, a fungal metabolite, and LY294002, a quercetin analog (Stack, J. H., and S. D. Emr. 1994. J. Biol. Chem. 269:31552-31562). We show here that at concentrations which inhibit VPS34-encoded PI 3-kinase activity, wortmannin also inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in mammalian cells with half-maximal inhibition of delivery occurring at 100 nM wortmannin. As a result of wortmannin action, newly synthesized, unprocessed cathepsin D is secreted into the media. Moreover, after accumulation in the trans-Golgi network (TGN) at 20 degrees C, cathepsin D was rapidly missorted to the secretory pathway after addition of wortmannin and shifting to 37 degrees C. At concentrations that inhibited lysosomal enzyme delivery, both wortmannin and LY294002 caused a highly specific dilation of mannose 6-phosphate receptor (M6PR)-enriched vesicles of the prelysosome compartment (PLC), which swelled to approximately 1 micron within 15 min after treatment. With increasing time, the inhibitors caused a significant yet reversible change in M6PR distribution. By 3 h of treatment, the swollen PLC vacuoles were essentially depleted of receptors and, in addition, there was a fourfold loss of receptors from the cell surface. However, M6PRs were still abundant in the TGN. These results are most consistent with the interpretation that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering with a M6PR-dependent sorting event in the TGN. Moreover, they provide evidence that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles rely on similar regulatory mechanisms.

Related Genes
VPS34
MeSH Terms
Acridine Orange/metabolism Androstadienes/pharmacology Animals Biological Transport Cathepsin D/metabolism Cell Compartmentation/physiology Cells, Cultured Chromones/pharmacology Enzyme Inhibitors/pharmacology Fluorescent Antibody Technique Golgi Apparatus/metabolism Hydrolases/metabolism Lysosomes/enzymology,ultrastructure Models, Biological Morpholines/pharmacology Phosphatidylinositol 3-Kinases Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors,metabolism Protein Processing, Post-Translational Receptor, IGF Type 2/metabolism Vacuoles/metabolism,ultrastructure Wortmannin
Chemicals
Androstadienes Chromones Enzyme Inhibitors Morpholines Receptor, IGF Type 2 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one Phosphatidylinositol 3-Kinases Phosphotransferases (Alcohol Group Acceptor) Hydrolases Cathepsin D Acridine Orange Wortmannin
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Brown W J
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
DeWald D B
Emr S D
Plutner H
Balch W E
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.130.4.781
Published
1995-08-00
Pages
781-96
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2199950
Subset
IM
Grants
NCI NIH HHS · CA58689 · United States
NIDDK NIH HHS · DK37249 · United States
NIGMS NIH HHS · GM42336 · United States
External Links
PubMed source DOI: 10.1083/jcb.130.4.781 PMC Full Text View journal details
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