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Vol. 15, Issue 7, 3132-3145, July 2004
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* Institute of Biochemistry, University of Kiel, D-24098 Kiel, Germany;
|| Institute of Anatomy, University of Kiel, D-24098 Kiel, Germany;
Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan;
# IBM II, Molecular Cell Biology, University Hospital Hamburg-Eppendorf, D-20246 Hamburg, Germany;
¶ Department for Human Genetics CB4, K.U. Leuven, and Flanders Interuniversity Institute for Biotechnology VIB IV, B-3000 Leuven, Belgium;
@ Department of Biochemistry 2, D-37073 Göttingen, Germany; and
Cell Biology, Instituto de Investigaciones Citológicas, FVIB, 46010 Valencia, Spain
Submitted February 5, 2004;
Revised April 13, 2004;
Accepted April 15, 2004
Monitoring Editor: Juan S. Bonifacino
Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.
Abbreviations used: Avi, early autophagic vacuole; Avd, late autophagic vacuole; BSA, bovine serum albumin; ER, endoplasmic reticulum; FCS, fetal calf serum; GFP, green fluorescent protein; HRP, horseradish peroxidase; LAMP, lysosomal associated membrane protein; LIMP, lysosomal integral membrane protein; LBPA, lysobisphosphatidic acid; LDL, low-density lipoprotein; LPDS, lipoprotein-deficient serum; 3MA, 3-methyladenine; MEF, mouse embryonic fibroblast; MPR, mannose-6-phosphate receptor; PBS, phosphate-buffered saline; TGN, trans-Golgi network.
Online version of this article contains supporting material. Online version is available at www.molbiolcell.org.
These authors contributed equally to this work.
** Corresponding author. E-mail address: psaftig{at}biochem.uni-kiel.de.
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