A 73-kilodalton (kD) intracellular protein was found to bind to peptide regions that target intracellular proteins for lysosomal degradation in response to serum withdrawal. This protein cross-reacted with a monoclonal antibody raised to a member of the 70-kD heat shock protein (hsp70) family, and sequences of two internal peptides of the 73-kD protein confirm that it is a member of this family. In response to serum withdrawal, the intracellular concentration of the 73-kD protein increased severalfold. In the presence of adenosine 5'-triphosphate (ATP) and MgCl2, the 73-kD protein enhanced protein degradation in two different cell-free assays for lysosomal proteolysis.

Lysosomal uptake and degradation of polypeptides such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribonuclease A (RNase A), and RNase S-peptide (residues 1-20 of RNase A) are progressively activated in rat liver by starvation before isolation of lysosomes. This pathway of proteolysis is selective, since it is stimulated by the heat shock cognate protein of 73 kDa (HSC73) and ATP-MgCl2, and lysosomal uptake of RNase A could be competed by GAPDH but not by ovalbumin. A portion of intracellular HSC73 is associated with certain lysosomes, and the amount of lysosomal HSC73 increases by 5- to 10-fold during prolonged starvation. The lysosome-associated HSC73 is primarily within the lysosomal lumen. Double immunogold labeling of lysosomes incubated in vitro with RNase A detects this protein substrate as well as HSC73 within lysosomes. More than two-thirds of the labeled lysosomes contain both RNase A and HSC73. The possible physiological significance of the activation of this selective pathway of lysosomal proteolysis in long-term starvation is discussed.

Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules.Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.

Peroxisomes are capable of reactive oxygen species (ROS) generation, but their contribution to cellular redox balance is not well understood. This study demonstrates that peroxisomes and mitochondria functionally interact via ROS signaling, suggesting a potential broader role for the peroxisome in cellular aging and the initiation and progression of age-related diseases.

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging