Cloning, expression, and cellular localization of a human prenylcysteine lyase

Prenylated proteins contain either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus. These proteins constitute up to 2% of total cellular protein in eukaryotic cells. The degradation of prenylated proteins raises a metabolic challenge to the cell, because the thioether bond of the modified cysteine is quite stable. We recently identified and isolated an enzyme termed prenylcysteine lyase that cleaves the prenylcysteine to free cysteine and an isoprenoid product (Zhang, L., Tschantz, W. R., and Casey, P. J. (1997) J. Biol. Chem. 272, 23354-23359). To facilitate the molecular characterization of this enzyme, its cloning was undertaken. Overlapping cDNA clones encoding the complete coding sequence of this enzyme were obtained from a human cDNA library. The open reading frame of the gene encoding prenylcysteine lyase is 1515 base pairs and has a nearly ubiquitous expression pattern with a message size of 6 kilobase pairs. Recombinant prenylcysteine lyase was produced in a baculovirus-Sf9 expression system. Analysis of both the recombinant and native enzyme revealed that the enzyme is glycosylated and contains a signal peptide that is cleaved during processing. Additionally, the subcellular localization of this enzyme was determined to be lysosomal. These findings strengthen the notion that prenylcysteine lyase plays an important role in the final step in the degradation of prenylated proteins and will allow further physiological and biochemical characterization of this enzyme.     

Lysosomal prenylcysteine lyase is a FAD-dependent thioether oxidase

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached via a thioether bond to a cysteine residue at or near their C terminus. As prenylated proteins comprise up to 2% of the total protein in eukaryotic cells, and the thioether bond is a stable modification, their degradation raises a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that cleaves prenylcysteines to cysteine and an unidentified isoprenoid product. Here we show that the isoprenoid product of prenylcysteine lyase is the C-1 aldehyde of the isoprenoid moiety (farnesal in the case of C-15). The enzyme requires molecular oxygen as a cosubstrate and utilizes a noncovalently bound flavin cofactor in an NAD(P)H-independent manner. Additionally, a stoichiometric amount of hydrogen peroxide is produced during the reaction. These surprising findings indicate that prenylcysteine lyase utilizes a novel oxidative mechanism to cleave thioether bonds and provide insight into the unique role this enzyme plays in the cellular metabolism of prenylcysteines.     

Proteomic analysis of human low‐density lipoprotein reveals the presence of prenylcysteine lyase,a hydrogen peroxide‐generating enzyme

The molecular mechanisms underlying the relationship between low‐density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid‐phase IEF has been used to resolve LDL proteins into well‐defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL‐associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol‐binding protein, and paraoxonase‐1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time‐dependent generation of H₂O₂ which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H₂O₂ generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis.     

Stereospecificity and kinetic mechanism of human prenylcysteine lyase,an unusual thioether oxidase

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus. The cellular abundance of prenylated proteins, as well as the stability of the thioether bond, poses a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that degrades a variety of prenylcysteines. Prenylcysteine lyase is a FAD-dependent thioether oxidase that produces free cysteine, an isoprenoid aldehyde, and hydrogen peroxide as products of the reaction. Here we report initial studies of the kinetic mechanism and stereospecificity of this unusual enzyme. We utilized product and dead end inhibitors of prenylcysteine lyase to probe the kinetic mechanism of the multistep reaction. The results with these inhibitors, together with those of other experiments, suggest that the reaction catalyzed by prenylcysteine lyase proceeds through a sequential mechanism. The reaction catalyzed by the enzyme is stereospecific, in that the pro-S hydride of the farnesylcysteine is transferred to FAD to initiate the reaction. With (2R,1'S)-[1'-(2)H(1)]farnesylcysteine as a substrate, a primary deuterium isotope effect of 2 was observed on the steady state rate. However, the absence of an isotope effect on an observed pre-steady-state burst of hydrogen peroxide formation implicates a partially rate-determining proton transfer after a relatively fast C-H (C-D) bond cleavage step. Furthermore, no pre-steady-state burst of cysteine was observed. The finding that the rate of cysteine formation was within 2-fold of the steady-state k(cat) value indicates that cysteine production is one of the primary rate-limiting steps in the reaction. These results provide substantial new information on the catalytic mechanism of prenylcysteine lyase.     

The crystal structure of palmitoyl protein thioesterase-2 (PPT2) reveals the basis for divergent substrate specificities of the two lysosomal thioesterases,PPT1 and PPT2

Mutations in palmitoyl protein thioesterase-1 (PPT1) have been found to cause the infantile form of neuronal ceroid lipofuscinosis, which is a lysosomal storage disorder characterized by impaired degradation of fatty acid-modified proteins with accumulation of amorphous granular deposits in cortical neurons, leading to mental retardation and death. Palmitoyl protein thioesterase-2 (PPT2) is a second lysosomal hydrolase that shares a 26% identity with PPT1. A previous study had suggested that palmitoyl-CoA was the preferred substrate of PPT2. Furthermore, PPT2 did not hydrolyze palmitate from the several S-palmitoylated protein substrates. Interestingly, PPT2 deficiency in a recent transgenic mouse model is associated with a form of neuronal ceroid lipofuscinosis, suggesting that PPT1 and -2 perform non-redundant roles in lysosomal thioester catabolism. In the current paper, we present the crystal structure of PPT2 at a resolution of 2.7 A. Comparisons of the structures of PPT1 and -2 show very similar architectural features; however, conformational differences in helix alpha4 lead to a solvent-exposed lipid-binding groove in PPT1. The limited space between two parallel loops (beta3-alphaA and beta8-alphaF) located immediately above the lipid-binding groove in PPT2 restricts the binding of fatty acids with bulky head groups, and this binding groove is significantly larger in PPT1. This structural difference accounts for the ability of PPT2 to hydrolyze an unbranched structure such as palmitoyl-CoA but not palmitoylcysteine or palmitoylated proteins. Furthermore, differences in fatty acid chain length specificity of PPT1 and -2, also reported here, are explained by the structure and may provide a biochemical basis for their non-redundant roles.     

Prenylcysteine methylesterase in Arabidopsis thaliana

Prenylated proteins undergo a series of post-translational modifications, including prenylation, proteolysis, and methylation. Collectively, these modifications generate a prenylcysteine methylester at the carboxyl terminus and modulate protein targeting and function. Prenylcysteine methylation is the only reversible step in this series of modifications. However, prenylcysteine -carboxyl methylesterase (PCME) activity has not been described in plants. We have detected a specific PCME activity in Arabidopsis thaliana membranes that discriminates between biologically relevant and irrelevant prenylcysteine methylester substrates. Furthermore, we have identified an Arabidopsis gene (At5g15860) that encodes measurable PCME activity in recombinant yeast cells with greater specificity for biologically relevant prenylcysteine methylesters than the activity found in Arabidopsis membranes. These results suggest that specific and non-specific esterases catalyze the demethylation of prenylcysteine methylesters in Arabidopsis membranes. Our findings are discussed in the context of prenylcysteine methylation/demethylation as a potential regulatory mechanism for membrane association and function of prenylated proteins in Arabidopsis.     

Protein acyl thioesterases (Review)

Many proteins are S-acylated, affecting their localization and function. Dynamic S-acylation in response to various stimuli has been seen for several proteins in vivo. The regulation of S-acylation is beginning to be elucidated. Proteins can autoacylate or be S-acylated by protein acyl transferases (PATs). Deacylation, on the other hand, is an enzymatic process catalyzed by protein thioesterases (APT1 and PPT1) but only APT1 appears to be involved in the regulation of the reversible S-acylation of cytoplasmic proteins seen in vivo. PPT1, on the other hand, is involved in the lysosomal degradation of S-acylated proteins and PPT1 deficiency causes the disease infant neuronal ceroid lipofuscinosis.     

Characterization of lipid-linked oligosaccharide accumulation in mouse models of Batten disease

The neuronal ceroid lipofuscinoses (NCLs, also known collectively as Batten disease) are a group of lysosomal storage disorders characterized by the accumulation of autofluorescent storage material in the brain and other tissues. A number of genes underlying various forms of NCL have been cloned, but the basis for the neurodegeneration in any of these is unknown. High levels of dolichol pyrophosphoryl oligosaccharides have previously been demonstrated in brain tissue from several NCL patients, but the specificity of the effect for the NCLs has been unclear. In the present study, we examine eight mouse models of lysosomal storage disorders by modern FACE and found striking lipid-linked oligosaccharide (LLO) accumulation in NCL mouse models (especially CLN1, CLN6, and CLN8 knockout or mutant mice) but not in several other lysosomal storage disorders affecting the brain. Using a mouse model of the most severe form of NCL (the PPT1 knockout mouse), we show that accumulated LLOs are not the result of a defect in LLO synthesis, extension, or transfer but rather are catabolic intermediates derived from LLO degradation. LLOs are enriched about 60-fold in the autofluorescent storage material purified from PPT1 knockoutmouse brain but comprise only 0.3% of the autofluorescent storage material by mass. The accumulation of LLOs is postulated to result from inhibition of late stages of lysosomal degradation of autophagosomes, which may be enriched in these metabolic precursors.     

Arabidopsis thaliana plants possess a specific farnesylcysteine lyase that is involved in detoxification and recycling of farnesylcysteine

In plants, prenylated proteins are involved in actin organization, calcium-mediated signal transduction, and many other biological processes. Arabidopsis thaliana mutants lacking functional protein prenyltransferase genes have also revealed roles for prenylated proteins in phytohormone signaling and meristem development. However, to date, the turnover of prenylated plant proteins and the fate of the prenylcysteine (PC) residue have not been described. We have detected an enzyme activity in Arabidopsis plants that metabolizes farnesylcysteine (FC) to farnesal, which is subsequently reduced to farnesol. Unlike its mammalian ortholog, Arabidopsis FC lyase exhibits specificity for FC over geranylgeranylcysteine (GGC), and recognizes N-acetyl-FC (AFC). FC lyase is encoded by a gene on chromosome 5 of the Arabidopsis genome (FCLY, At5g63910) and is ubiquitously expressed in Arabidopsis tissues and organs. Furthermore, T-DNA insertions into the FCLY gene cause significant decreases in FC lyase activity and an enhanced response to abscisic acid (ABA) in seed germination assays. The effects of FCLY mutations on ABA sensitivity are even greater in the presence of exogenous FC. These data suggest that plants possess a specific FC detoxification and recycling pathway.     

Hyperthermia stimulates energy-proteasome-dependent protein degradation in cultured myotubes

Previous studies suggest that elevated temperature stimulates protein degradation in skeletal muscle, but the intracellular mechanisms are not fully understood. We tested the role of different proteolytic pathways in temperature-dependent degradation of long- and short-lived proteins in cultured L6 myotubes. When cells were cultured at different temperatures from 37 to 43 degrees C, the degradation of both classes of proteins increased, with a maximal effect noted at 41 degrees C. The effect of high temperature was more pronounced on long-lived than on short-lived protein degradation. By using blockers of individual proteolytic pathways, we found evidence that the increased degradation of both long-lived and short-lived proteins at high temperature was independent of lysosomal and calcium-mediated mechanisms but reflected energy-proteasome-dependent degradation. mRNA levels for enzymes and other components of different proteolytic pathways were not influenced by high temperature. The results suggest that hyperthermia stimulates the degradation of muscle proteins and that this effect of temperature is regulated by similar mechanisms for short- and long-lived proteins. Elevated temperature may contribute to the catabolic response in skeletal muscle typically seen in sepsis and severe infection.     

The carboxyl methyltransferase modifying G proteins is a metalloenzyme.

The prenylated protein carboxyl methyltransferase (PPMT) catalyzes the posttranslational methylation of isoprenylated C-terminal cysteine residues found in many signaling proteins such as the small monomeric G proteins and the gamma subunits of heterotrimeric G proteins. Here we report that both membrane-bound PPMT from rat kidney and the recombinant bacterially expressed form of the enzyme required divalent cations for catalytic activity. Unlike EDTA and EGTA, the metal chelator 1,10-phenanthroline strongly inhibited the PPMT activity of kidney intracellular membranes in a dose- and time-dependent manner. 1,10-Phenanthroline was found to inhibit the methylation of the prenylcysteine analog N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, a synthetic substrate for PPMT, with an IC(50) of 2.2 mM. Gel electrophoretic analysis demonstrated that 1,10-phenanthroline almost totally abolished the labeling of methylated proteins in kidney intracellular membranes. Immunoblotting analysis showed that one of the two major peaks of (3)H-methylated proteins in intracellular membranes comigrated with the small G proteins Ras, Cdc42, RhoA, and Rab1. In addition, the methylation of immunoprecipitated Ras and RhoA from kidney intracellular membranes was strongly inhibited when 1,10-phenanthroline was present. Treatment of kidney intracellular membranes with 1,10-phenanthroline increased the proteolytic degradation of PPMT by exogenous trypsin, compared to untreated membranes. We conclude from these data that metal ions are essential for the activity and the stabilization of PPMT. The finding that PPMT is a metalloenzyme may provide new insights into the functions played by this methyltransferase in signal transduction processes.     

Human palmitoyl protein thioesterase: evidence for lysosomal targeting of the enzyme and disturbed cellular routing in infantile neuronal ceroid lipofuscinosis.

Palmitoyl protein thioesterase (PPT) is an enzyme that removes palmitate residues from various S-acylated proteins in vitro. We recently identified mutations in the human PPT gene in patients suffering from a neurodegenerative disease in childhood, infantile neuronal ceroid lipofuscinosis (INCL), with dramatic manifestations limited to the neurons of neocortical origin. Here we have expressed the human PPT cDNA in COS-1 cells and demonstrate the lysosomal targeting of the enzyme via the mannose 6-phosphate receptor-mediated pathway. The enzyme was also secreted into the growth medium and could be endocytosed by recipient cells. We further demonstrate the disturbed intracellular routing of PPT carrying the worldwide most common INCL mutation, Arg122Trp, to lysosomes. The results provide evidence that INCL represents a novel lysosomal enzyme deficiency. Further, the defect in the PPT gene causing a neurodegenerative disorder suggests that depalmitoylation of the still uncharacterized substrate(s) for PPT is critical for postnatal development or maintenance of cortical neurons.     

Chasing the elusive mammalian microautophagy

Different mechanisms for delivery of intracellular components (proteins and organelles) to lysosomes and late endosomes for degradation co-exist in almost all cells and set the basis for distinct autophagic pathways. Cargo can be sequestered inside double-membrane vesicles (or autophagosomes) and reach the lysosomal compartment upon fusion of these vesicles to lysosomes through macroautophagy. In a different type of autophagy, known as chaperone-mediated autophagy (CMA), single individual soluble proteins can be targeted one by one to the lysosomal membrane and translocated into the lumen for degradation. Direct sequestration of proteins and organelles by invaginations at the lysosomal membrane that pinch off into the lumen has also been proposed. This process, known as microautophagy, remains poorly understood in mammalian cells. In our recent work, we demonstrate the occurrence of both "in bulk" and "selective" internalization of cytosolic components in late endosomes and identify some of the molecular players of this process that we have named endosomalmicroautophagy (e-MI) due to its resemblance to microautophagy.     

Towards complete sets of farnesylated and geranylgeranylated proteins

Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein-protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS) has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase) and can be queried for verification status, type of modifying enzymes (anchor type), and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins-for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain-containing FLJ32421 (termed BROFTI), and Rab28 (short isoform) as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for the selective farnesylation of targets with an evolutionary conserved modification site.     

Cloning and characterization of a mammalian prenyl protein-specific protease

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Ggamma1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.     

Storage problems in lysosomal diseases

Biochemical disorders in lysosomal storage diseases consist of the interruption of metabolic pathways involved in the recycling of the degradation products of one or several types of macromolecules. The progressive accumulation of these primary storage products is the direct consequence of the genetic defect and represents the initial pathogenic event. Downstream consequences for the affected cells include the accumulation of secondary storage products and the formation of histological storage lesions, which appear as intracellular vacuoles that represent the pathological hallmark of lysosomal storage diseases. Relationships between storage products and storage lesions are not simple and are still largely not understood. Primary storage products induce malfunction of the organelles where they accumulate, these being primarily, but not only, lysosomes. Consequences for cell metabolism and intracellular trafficking combine the effects of primary storage product toxicity and the compensatory mechanisms activated to protect the cell. Induced disorders extend far beyond the primarily interrupted metabolic pathway.     

A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate its membrane proteins is poorly understood. In this study, we performed a cycloheximide (CHX) chase assay to measure the half-lives of approximately 30 human lysosomal membrane proteins (LMPs) and identified RNF152 and LAPTM4A as short-lived membrane proteins. The degradation of both proteins is ubiquitin dependent. RNF152 is a transmembrane E3 ligase that ubiquitinates itself, whereas LAPTM4A uses its carboxyl-terminal PY motifs to recruit NEDD4-1 for ubiquitination. After ubiquitination, they are internalized into the lysosome lumen by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation. Strikingly, when ectopically expressed in budding yeast, human RNF152 is still degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

A study of how lysosomal membrane proteins are down-regulated reveals a conserved pathway involving ubiquitination of the membrane protein and subsequent internalization into the lysosome lumen by the ESCRT machinery for degradation.     

Mycobacterium tuberculosis Type VII Secreted Effector EsxH Targets Host ESCRT to Impair Trafficking

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D''Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.     

Microinjected ribonuclease A as a probe for lysosomal pathways of intracellular protein degradation

There are multiple pathways of intracellular protein degradation, and molecular determinants within proteins appear to target them for particular pathways of breakdown. We use red cell-mediated microinjection to introduce radiolabeled proteins into cultured human fibroblasts in order to follow their catabolism. A well-characterized protein, bovine pancreatic ribonuclease A (RNase A), is localized initially in the cytosol of cells after microinjection, but it is subsequently taken up and degraded by lysosomes. This lysosomal pathway of proteolysis is subject to regulation in that RNase A is taken up and degraded by lysosomes at twice the rate when serum is omitted from the culture medium. Subtilisin cleaves RNase A between residues 20 and 21, and the separated fragments are termed RNase S-peptide (residues 1–20) and RNase S-protein (residues 21–124). Microinjected RNase S-protein is degraded in a serum-independent manner, while RNase S-peptide microinjected alone shows a twofold increase in degradation in response to serum withdrawal. Furthermore, covalent linkage of S-peptide to other proteins prior to microinjection causes degradation of the conjugate to become serum responsive. These results show that recognition of RNase A and certain other proteins for enhanced lysosomal degradation during serum withdrawal is based on some feature of the amino-terminal 20 amino acids. The entire S-peptide is not required for enhanced lysosomal degradation during serum withdrawal because degradation of certain fragments is also responsive to serum. We have identified the essential region to be within residues 7–11 of RNase S-peptide (Lys-Phe-Glu-Arg-Gln; KFERQ). To determine whether related peptides exist in cellular proteins, we raised antibodies to the pentapeptide. Affinity-purified antibodies to KFERQ specifically precipitate 25–35% of cellular proteins, and these proteins are preferentially degraded in response to serum withdrawal. Computer analyses of known protein sequences indicate that proteins degraded by lysosomes at an enhanced rate in response to serum withdrawal contain peptide regions related, but not identical, to KFERQ. We suggest two possible peptide motifs related to KFERQ and speculate about possible mechanisms of selective delivery of proteins to lysosomes based on such peptide regions.