Ubiquitinated protein conjugates are specifically enriched in the lysosomal system of fibroblasts

Abstract

Ubiquitin-protein conjugates are found by immunogold electron microscopy to be enriched (12-fold) in the lysosomal compartment of 3T3-Ll fibroblasts. Treatment of fibroblasts with the cysteine protease inhibitor E-64 leads to an expansion of the lysosomal compartment and as a result an increase in the cellular content of ubiquitin-protein conjugates. There is no change in the specific enrichment of ubiquitin-protein conjugates in the lysosomal compartment following E-64 treatment. The results suggest that some ubiquitin-protein conjugates may normally be degraded lysosomally following sequestration by microautophagy and imply that protein ubiquitination may be one of the signals for protein uptake into lysosomes.

Immunogold localisation of ubiquitin‐protein conjugates in Sf9 insect cells

“…MVB-containing discrete areas of acid phosphatase Quantitation of gold particle densities in lysosome- related organelles (LVMC and MVB) relative to cytoplasm shows a comparable enrichment in 20-hydroxyecdysone treated cells and in control cells (Table I). These results are similar to our previous findings in fibroblasts [3,5], and indicate that the large volume expansion of the lysosome-related compartment by steroid action (approx. 4-fold) is accompanied by a similar increase in the concentration of ubiquitin-protein conjugates in the organelles.…”

“…UK), 2 mM CeCI, (Sigma), and 5% (w/v) sucrose [7]. Immunogold labelling was carried out by a post-embedding biotin-antibiotin bridge method [3,4] using an affinity-purified sheep antibody, which recognises ublquitin conjugated to other proteins [5,8], at a dilution of I:100 (nonimmune sheep IgG was applied for the controls at the same concentration) followed by biotmylated goat antibody to sheep IgG (Vector Labs.. Peterborough, UK; 1:200 dilution) and antibiotm-gold (IO nm, Bio-Rad, Hemel Hempstead. UK; 1:200 dilution).…”

Altered gene expression in cells from patients with lysosomal storage disorders suggests impairment of the ubiquitin pathway

“…This compound enters cells by pinocytosis and inhibits lysosomal protein degradation, causing a significant increase in the number and volume of lysosomes as well as the formation of multivesicular dense bodies. 22,23 In our experiments, 16-h incubation of normal control fibroblasts in the medium containing 0.5 mM E-64 significantly increased lysosome number and size as visualized by immunohistochemical staining with anti-LAMP-2 antibodies (Figure 2a). The same organelles were also heavily stained with antiubiquitin antibodies showing the accumulation of ubiquitinated proteins in the lysosomes ( Figure 2b).…”

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“…involved in autophagic protein degradation. Thus, in fibroblasts ubiquitin-protein conjugates can be found in the lysosomes, as shown by immunohistochemistry and immunogold electron microscopy (Doherty et al, 1989;Laszlo et al, 1990). Free ubiquitin can also be found inside lysosomes (Schwartz et al, 1988).…”