An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation.

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.

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.

A receptor for the selective uptake and degradation of proteins by lysosomes.

Multiple pathways of protein degradation operate within cells. A selective protein import pathway exists for the uptake and degradation of particular cytosolic proteins by lysosomes. Here, the lysosomal membrane glycoprotein LGP96 was identified as a receptor for the selective import and degradation of proteins within lysosomes. Specific substrates of this proteolytic pathway bound to the cytosolic tail of a 96-kilodalton lysosomal membrane protein in two different binding assays. Overexpression of human LGP96 in Chinese hamster ovary cells increased the activity of the selective lysosomal proteolytic pathway in vivo and in vitro.

A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins.

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.

Peptide sequences that target cytosolic proteins for lysosomal proteolysis.

Lysosomes take up and degrade intracellular proteins in cultured cells in response to serum deprivation, and in tissues of organisms in response to starvation. One mechanism by which proteins enter lysosomes for subsequent degradation requires that substrate proteins contain peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ).

IkappaB is a substrate for a selective pathway of lysosomal proteolysis.

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor kappa B (IkappaB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IkappaB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IkappaB is directly transported into isolated lysosomes in a process that requires binding of IkappaB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IkappaB uptake by lysosomes. Ubiquitination and phosphorylation of IkappaB are not required for its targeting to lysosomes. The lysosomal degradation of IkappaB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IkappaB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IkappaB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IkappaB is increased. There are both short- and long-lived pools of IkappaB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IkappaB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IkappaB degradation in lysosomes. This selective pathway of lysosomal degradation of IkappaB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor kappa B. In addition, the response of nuclear factor kappa B to several stimuli increases when this lysosomal pathway of proteolysis is activated.

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.

Turnover of F1F0-ATP synthase subunit 9 and other proteolipids in normal and Batten disease fibroblasts.

Fibroblasts derived from patients with late infantile neuronal ceroid lipofucsinosis (NCL) and from a mouse model of NCL are similar to cells in intact animals in that they accumulate subunit 9 of mitochondrial F1F0-ATP synthase (F-ATPase) (Tanner, A., Dice, J.F., Cell Biol. Int. 19 (1995) 71-75). We now report no differences in the synthetic rates of F-ATPase subunit 9 in such affected cells when compared to control cells. However, the degradation rates of F-ATPase subunit 9 are reduced in both the affected human and mouse cells. This reduced degradation applies only to subunit 9 and the homologous vacuolar ATPase subunit among five distinct, reproducible proteolipid bands analyzed. Approximately 15% of newly synthesized F-ATPase subunit 9 is rapidly degraded in control cells, but this rapidly degraded component is absent in both the human and mouse NCL fibroblasts. At confluence, when the accumulated F-ATPase subunit 9 transiently disappears from human NCL fibroblasts, there is an increased degradation of all proteolipids. The pathway of degradation that is enhanced at confluence is likely to correspond to lysosomal macroautophagy. We confirmed that lysosomes were able to degrade F-ATPase subunit 9 after endocytosis of radiolabeled mitochondria. Human NCL fibroblasts were less active than control cells in this lysosomal degradation of endocytosed F-ATPase subunit 9. However, this difference was not specific for F-ATPase subunit 9 since it also applied to total endocytosed mitochondrial protein. We conclude that degradation of F-ATPase subunit 9 can occur by multiple pathways and that a mitochondrial pathway of proteolysis is defective in the late infantile human and mouse forms of NCL.

Lysosomes, a meeting point of proteins, chaperones, and proteases.

Lysosomes, classically considered as nonspecific systems for protein degradation, have recently also been shown to be able selectively to degrade specific intracellular proteins. Here we review this selective pathway of lysosomal protein degradation that involves cytosolic and intralysosomal chaperones and a receptor in the lysosomal membrane. This pathway is highly selective for cytosolic proteins containing a lysosomal targeting signal. The selective lysosomal degradation pathway is active under conditions of nutrient deprivation and plays an important role in the regulation of intracellular protein levels in stress situations. Several physiological and pathological modifications in the activity of this selective lysosomal pathway of protein degradation are discussed.

Peptide signals for protein degradation within lysosomes.

In this article we summarize our findings concerning a pathway by which cytosolic proteins can be selectively taken up and degraded within lysosomes. Serum deprivation of cells in culture activates this pathway, and only proteins that contain peptide sequences related to KFERQ are degraded at an enhanced rate. Approximately 30% of intracellular proteins contain such peptide sequences, and we speculate about the physiological relevance of the selective degradation of these proteins in response to serum withdrawal. Several rat tissues also contain proteins with peptide sequences related to KFERQ, and the amount of these proteins is reduced in response to starvation. Finally, we present recent results suggesting that this selective uptake of cytosolic proteins by lysosomes is not through classical macroautophagy. Instead, the selective uptake appears to be similar to other protein sorting pathways such as protein translocation through the endoplasmic reticulum or protein import into mitochondria.

Altered degradation of intracellular proteins in aging human fibroblasts.

Confluent cultures of fibroblasts at different population doubling levels were incubated with [14C]leucine for 2 days and with [3H]leucine for 2 h to label long-lived and short-lived proteins, respectively. Proteolysis was then measured in the presence of excess unlabeled leucine to prevent reutilization of the isotope. Catabolism of long-lived proteins was reduced in senescent cells when measured in media without fetal bovine serum, insulin, fibroblast growth factor, or dexamethasone. In contrast, degradation of short-lived proteins was increased in senescent cells but only when measured in the presence of serum, hormones, and growth factors. Further experiments with cells of varying ages indicate that in unsupplemented medium half-lives of long-lived proteins lengthened by as much as 20 min per population doubling and in supplemented media half-lives of short-lived proteins decreased by 4 min per population doubling. The reduced catabolism of long-lived proteins in senescent cells cannot be explained by age-related changes in protein secretion or cell death during degradation measurements. These alterations in proteolysis may have major effects on protein content and composition in senescent cells.

How do intracellular proteolytic systems change with age?

One of the common features of cells from senescent tissues is the accumulation of abnormal proteins. Several hypotheses have been proposed to explain the origin of those abnormal proteins. A defect in proteolytic systems usually responsible for the elimination of altered proteins from the cells could clearly contribute to such accumulation. Here we describe the effect of age on the major proteolytic systems within cells: the ubiquitin-proteasome pathway, the calcium-activated calpain pathways, and multiple lysosomal pathways. Our group has contributed to the characterization of a selective pathway of degradation of cytosolic proteins in lysosomes that is activated under conditions of nutrient deprivation. In this lysosomal pathway of proteolysis proteins are transported through the lysosomal membrane assisted by cytosolic and lysosomal molecular chaperones and a receptor protein in the lysosomal membrane. The activity of this pathway significantly decreases with age, and this decrease might account for the cytosolic accumulation of aberrant substrate proteins in senescent cells. The cellular consequences of the decline of this lysosomal pathway together with possible methods to restore the reduced function are also addressed in this review.

Targeting of cytosolic proteins to lysosomes for degradation.

We review evidence for a pathway by which specific cytosolic proteins are targeted to lysosomes for degradation in cultured cells in response to serum withdrawal. This pathway is also activated by starvation in several rat tissues. The enhanced degradation is specific for a class of intracellular proteins containing peptide sequences related to residues 7 to 11 of ribonuclease A (RNase A). The amino acid sequence of this pentapeptide is lysine-phenylalanine-glutamate-arginine-glutamine, or, in single letter amino acid abbreviations, KFERQ. A heat shock protein of 73 kDa binds to such peptide regions in proteins and somehow mediates their transfer to lysosomes for degradation. The recent reconstitution of this lysosomal pathway of proteolysis in vitro should permit detailed mechanistic analysis of how proteins are directed to and translocated across lysosomal membranes.

Altered intracellular protein degradation in aging: a possible cause of proliferative arrest.

Many proteins that control cell-cycle progression are short-lived. Therefore, alterations in protein degradation are as likely as changes in transcription and/or translation in causing the proliferation arrest of senescent cells. Several different pathways of intracellular protein degradation have been identified, and both cytosolic and lysosomal pathways operate in most cells. We have used red cell-mediated microinjection to study degradation of radiolabelled proteins introduced into IMR-90 human diploid fibroblasts at early and late population doubling levels. Lysosomal pathways of protein degradation are reduced in senescent cells, and this defect may account for many characteristics of aging, including the accumulation of posttranslationally altered proteins. These abnormal proteins may then stimulate cytosolic, ubiquitin-dependent proteolytic pathways that are also responsible for the degradation of crucial regulatory proteins. Unknown short-lived proteins are also required for some step in lysosomal proteolysis, and this connection between the two degradative systems may cause the age-related changes in protein degradation to be progressive. Several experimental approaches are available to test whether altered protein degradation significantly contributes to proliferative arrest of senescent cells.

When lysosomes get old.

Changes in the lysosomes of senescent tissues and organisms are common and have been used as biomarkers of aging. Lysosomes are responsible for the degradation of many macromolecules, including proteins. At least five different pathways for the delivery of substrate proteins to lysosomes are known. Three of these pathways decline with age, and the molecular explanations for these deficiencies are currently being studied. Other aspects of lysosomal proteolysis increase or do not change with age in spite of marked changes in lysosomal morphology and biochemistry. Age-related changes in certain lysosomal pathways of proteolysis remain to be studied. This area of research is important because abnormalities in lysosomal protein degradation pathways may contribute to several characteristics and pathologies associated with aging.

Ubiquitin pools, ubiquitin mRNA levels, and ubiquitin-mediated proteolysis in aging human fibroblasts.

Senescent cells have less free ubiquitin and more ubiquitin-protein conjugates than do young cells. The ubiquitin-protein conjugates are heterogeneous in size but contain prominent bands at 106, 55, and 22 kDa. The age-related increase in ubiquitin-protein conjugates applies primarily to the 55-kDa band, while the 106-kDa and 22-kDa conjugates change little with age. Ubiquitin mRNA levels do not change with age, and the ability of cells to degrade two proteins that are good substrates for ubiquitin-mediated proteolysis is unaltered by aging. These results indicate that an increase in ubiquitin-protein conjugates does not necessarily reflect alterations in ubiquitin-mediated proteolysis. Furthermore, an overactive pathway of ubiquitin-mediated proteolysis does not appear to contribute to the proliferative arrest in senescent cells.

Selective degradation of cytosolic proteins by lysosomes.

Lysosomes are able to internalize cellular proteins in a variety of ways. One pathway is selective for cytosolic proteins containing peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ). This pathway is activated in confluent monolayers of cultured cells in response to deprivation of serum growth factors and applies to approximately 30% of cytosolic proteins. We have reconstituted this lysosomal degradation pathway in vitro. Uptake and/or degradation is stimulated by ATP and a member of the heat shock 70-kilodalton protein family, the 73-kilodalton constitutive heat shock protein. Several possible mechanisms of selective protein transport into lysosomes and the possible relevance of this proteolytic pathway to the processing of the amyloid precursor protein are discussed.

Microinjection of cultured cells using red-cell-mediated fusion and osmotic lysis of pinosomes: a review of methods and applications.

Proteins and other macromolecules can be injected into cultured cells by several different methods. Here we review the strengths and limitations of two of these methods, red-cell-mediated microinjection and osmotic lysis of pinosomes, and indicate how they may be successfully applied to the study of cultured cells.