Figure 3.

Accumulation of autophagic vacuoles in LAMP-1/LAMP-2 double-deficient MEFs. (A) Primary and SV-40 large T-antigen immortalized (CoSV, LA1/2-/-SV) MEFs were cultured in the presence or absence of serum and amino acids for 2 h. The amounts of early (Avi) and late autophagic vacuoles (Avd) were determined by quantitative electron microscopy. Co, control cells; LA1-/-, LAMP-1-deficient cells; LA2-/-, LAMP-2-deficient cells, LA1/2-/- double-deficient cells. The numbers (5, etc.) indicate individual cell lines. (B) Electron micrograph of LAMP-1/LAMP-2 double-deficient cells (79), which were fed with BSA gold in serum-free medium for 2 h to label endosomes and lysosomes. Early (AVi) and late autophagic vacuoles (AVd) were abundant. Multivesicular bodies (MVB) and late endosomes (LE) containing BSA gold are also indicated. Arrowheads indicate late autophagic vacuoles which have fused with BSA-gold positive endosomes. (C) Western blotting of the autophagic marker protein LC3 in nonstarved cells and in cells incubated in serum and amino acid free medium for 2 h. The locations of LC3 precursor (LC3prec.) as well as LC3I and LC3II are indicated on the right. (D) Quantitation of the absolute levels of LC3I and LC3II in the Western blot shown in C for control and double-deficient cells. The values represent the average and SD of cell lines with identical genotype. (E) LC3 Western blot of LAMP-1 and LAMP-2 single-deficient liver and heart extracts. (F) Degradation of long lived proteins in the absence or presence of 3-methyladenine (3MA). MEFs were metabolically labeled with [³H]valine or [³H]leucine and then chased for 24 h in full medium. Cells were then switched to culture medium with serum (20% FCS) or to serum and amino acid free medium (w/o AA) for 4 h. TCA-soluble and -precipitable radioactivities were measured from the culture medium. Protein degradation was measured as the net release of TCA-soluble radioactivity, expressed as percentage of the total TCA-precipitable radioactivity present in cells at the beginning of the incubation. The results are mean and SD from 9-14 (20% FCS) or 6 experiments (w/o AA) with 2-3 parallel samples. Three (control, LA1-/-, LA2-/-) or one (LA1/LA2-/-, 79) independent cell lines were used.