Turnover of plasma membrane proteins in rat hepatoma cells and primary cultures of rat hepatocytes

The half-lives of turnover of plasma membrane proteins in rat hepatoma tissue, culture cells, and in primary cultures of rat hepatocytes have been analyzed after resolution by two-dimensional gel electrophoresis. Cell membranes were externally labeled via iodination catalyzed by lactoperoxidase and glucose oxidase. A bimodal pattern of turnover was found for the externally oriented plasma membrane proteins of rat hepatoma cells. Three glycoproteins analyzed in these cells had an average t 1/2 of 22 h while eight proteins which did not bind to concanavalin A had an average t 1/2 of 80 h. In contrast, more heterogeneous rates of turnover were found for the externally oriented plasma membrane proteins of primary cultures of hepatocytes. Most, if not all, of the membrane proteins accessible to iodination in these cells were glycoproteins. Among the glycoproteins resolved by two-dimensional polyacrylamide electrophoresis, the receptors for asialoglycoproteins had the shortest half-lives (18 h). Other glycoproteins, mostly with higher molecular weights and different isoelectric points, showed a spectrum of half-lives ranging from 16 to 99 h. The turnover rates of membrane proteins of primary cultures of rat hepatocytes were also determined with [3H]- and [35S]methionine labeling of cells. Heterogeneous rates of turnover again were found among the labeled glycoproteins and nonglycoproteins. Among the 10 glycoproteins individually analyzed, the half-lives range from 17 to 67 h. Among the 21 proteins which do not bind to concanavalin A, the half-lives range from 18 h to more than 100 h. Three proteins analyzed showed an apparent biphasic pattern of turnover, having a fast phase with a half-life of 4-6 h and a slow phase with a half-life of 15-29 h. Several nonglycoproteins, including clathrin and actin associated with membrane vesicles had extremely long half-lives. The more than 5-fold difference in the half-life between clathrin and the receptors for asialoglycoproteins, which coexist in coated pits indicates that intrinsic proteins of the coated pits turn over at a different rate than peripheral components.     

Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.     

Temporal regulation of salmonella virulence effector function by proteasome-dependent protein degradation

Salmonella enterica invasion of host cells requires the reversible activation of the Rho-family GTPases Cdc42 and Rac1 by the bacterially encoded GEF SopE and the GAP SptP, which exert their function at different times during infection and are delivered into host cells by a type III secretion system. We found that SopE and SptP are delivered in equivalent amounts early during infection. However, SopE is rapidly degraded through a proteosome-mediated pathway, while SptP exhibits much slower degradation kinetics. The half-lives of these effector proteins are determined by their secretion and translocation domains. Chimeric protein analysis indicated that delivery of SptP into host cells by the SopE secretion and translocation domain drastically shortened its half-life. Conversely, delivery of SopE by the SptP secretion and translocation signals significantly increased its half-life, resulting in persistent actin cytoskeleton rearrangements. This regulatory mechanism constitutes a remarkable example of a pathogen's adaptation to modulate cellular functions.     

Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells

The turnover of myosin and actin in both muscle and non-muscle cells in culture was investigated. By the double-label criterion, myosin and actin were coordinately synthesized and degraded in replicating, mononucleated fibroblasts, chondrocytes, BUdR-suppressed myogenic cells, and in post-mitotic, multinucleated myotubes. Myosin and actin were among the most stable proteins in each cell type. In single label ‘pulse-chase’ experiments, the half-lives of myosin and actin in all replicating, mononucleated cells were 2.5–3 days; in myotubes, however, they were approx. 6 days. Myosin and actin labelled in replicating presumptive myoblasts and chased until the cells ceased replicating and fused into multinucleated myotubes retained the degradation rate of 3 days; this differed from Jhe rate of 6 days shown for myosin and actin newly-synthesized in post-mitotic myotubes. The type of myosin synthesized in the mother presumptive myoblast, then, is transmitted to the postmitotic daughters. This myosin, however, is more rapidly degraded than the definitive myosin that is synthesized in the myotube.     

Two-dimensional electrophoretic analyses of proteins synthesized during differentiation of 3T3-L1 preadipocytes.

The changes in protein composition and cell surface proteins that occur during the adipocyte conversion of 3T3-L1 preadipocytes were monitored by two-dimensional polyacrylamide gel electrophoresis folowing incubation of cells with [35S]methionine for periods of 3 and 24 h. Alterations in the biosynthesis of more than 30 cytoplasmic proteins, 9 non-histone, chromosome-associated proteins, and 24 membrane proteins, were detected. Although the methodological limitations of the electrophoretic systems employed result in an underestimate of the total number of differences, the alterations observed exceed the enzyme changes known to occur during differentiation of these cells. One major alteration occurring during differentiation is a decrease in the content of a protein whose position following two-dimensional electrophoresis tentatively identified it as actin. A fall in actin content accompanying adipocyte conversion was confirmed by direct analysis of the DNase 1 inhibitory activity in homogenates prepared from cells during the course of differentiation. Studies of cell surface proteins by lactoperoxidase-catalyzed iodination reveal a number of changes during differentiation including an increase in a polypeptide(s) in the molecular weight range of 16,500 to 18,500, a decrease in at least four proteins of molecular weights greater than 100,000, and in a protein of molecular weight 95,000.     

Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture

Addition of pepstatin, an inhibitor of acid protease, to 2-day cultures of rat hepatocytes rapidly inhibited the activity to hydrolyze hemoglobin (Hb), but did not affect the activity to hydrolyze α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). On the other hand, addition of leupeptin, an inhibitor of thiol protease, inhibited the activity of BANA hydrolase and caused a sixfold increase in the activity of Hb hydrolase within 1 day. Neither protease inhibitor affected the rate of protein synthesis. Release of amino acids from hepatocytes into Hanks' salt solution was measured by the ninhydrin method. Pepstatin inhibited the release only 15% within 2 days, but leupeptin inhibited it 65% within 10 h. These two inhibitors had additive inhibitory effects on the release, suggesting that they inhibit the degradations of different groups of proteins. The inhibitory effect of leupeptin gradually decreased after 10 h, which is consistent with the observed induction of a protease activity mentioned above. A preferential involvement of leupeptin-sensitive protease in the degradation of proteins with longer half-lives was suggested from studies on [¹⁴C]leucine release from hepatocytes prelabeled for 30 h. On the other hand, the two inhibitors had similar effects on the release of [¹⁴C]leucine from hepatocytes labeled for only 1 h. Their inhibitory effects were again additive, but there was no reduction in the inhibition by leupeptin on prolonged incubation, suggesting that proteins with short half-lives were not substrates for the induced protease. These results suggest that in hepatocytes, proteins with longer half-lives are degraded more by cathepsin B than by cathepsin D, while those with short half-lives are degraded equally by these two proteases.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.     

The diversity of protein turnover and abundance under nitrogen-limited steady-state conditions in Saccharomyces cerevisiae

To establish more advanced models of molecular dynamics within cells, protein characteristics such as turnover rate and absolute instead of relative abundance have to be analyzed. We applied a proteomics strategy to analyze protein degradation and abundance in Saccharomyces cerevisiae. We used steady-state chemostat cultures to ascertain well-defined growth conditions and nitrogen limited media, which allowed us to rapidly switch from (14)N to (15)N-isotope containing media and to monitor the decay of the (14)N mono-isotope signals in time. We acquired both protein abundance information and degradation rates of 641 proteins. Half-lives of individual proteins were very diverse under nitrogen-limited steady-state conditions, ranging from less than 30 min to over 20 h. Proteins that act as single physical complexes do not always show alike half-lives. For example the chaperonin-containing TCP-1 complex showed similar intermediate half-lives ranging from 7 to 20 h. In contrast, the ribosome exhibited a wide diversity of half-lives ranging from 2.5 to over 20 h, although their cellular abundances were rather similar. The stabilities of proteins involved in the central sugar metabolism were found to be intermediary, except for the glycolytic enzymes Hxk1p and Fba1p and the TCA-cycle proteins Lsc2p and Kgd1p, which showed half-lives of over 20 h. These data stress the need for inclusion of quantitative data of protein turn-over rates in yeast systems biology.     

Isolation and characterization of soybean waste-degrading microorganisms and analysis of fertilizer effects of the degraded products.

Two microorganisms which could degrade soybean lees efficiently were isolated and identified as Bacillus circulans and B. stearothermophilus. These two strains secreted thermostable proteases into the medium and could digest soybean lees rapidly and completely at 50 degrees C. Initially, the soybean lees were degraded to proteins in approximately 20 h by these two strains, after which time the concentrations of peptides in the medium gradually increased. The degraded products from soybean lees contained abundant nitrogen compounds, such as peptides, amino acids, and amides. Approximately 10 times more fresh plant weight was obtained (in the case of Brassica campestris) when these degraded products were applied than when water was applied for 42 days. These stimulatory effects of the soybean lees products were almost equal to those of a chemically synthesized fertilizer.     

Bovine viral diarrhea virus proteins: relatedness of p175 with p80 and p125 and evidence of glycoprotein processing.

Three monoclonal antibodies, which recognized two nonoverlapping antigenic domains and were reactive to the bovine viral diarrhea virus (BVDV) p80 protein, were found to cross react with the p125 protein of both cytopathic and noncytopathic BVDVs and a molecular weight 175,000 BVDV protein (p175). Results from limited proteolysis and chemical cleavage experiments confirmed the relatedness of these three proteins. In pulse-chase experiments it was apparent that p175 was a transient protein, as it was diminished during the chase, with a half-life of about 30 min. However, both p125 and p80 were also observed in short-pulsed lysates. Furthermore, during the chase, radiolabel was not found to accumulate into p125 or p80. Rather, these two proteins were stable with half-lives greater than 2 h. A fourth nonglycosylated protein, p37, increased during the chase. Processing of several glycoproteins was evident in these experiments. A glycoprotein of molecular weight 75,000 (gp75) diminished during the chase period, while glycoproteins gp62, gp48, and gp25 appeared or increased during the chase period. In contrast, the glycoprotein gp53 was a major protein in pulse-labeled cell lysates and remained constant throughout the chase period. In further experiments two stable forms of p80 differing in intramolecular disulphide bonding were observed.     

Turnover of the creatine kinase subunits in chicken myogenic cell cultures and in fibroblasts.

The rates of degradation of creatine kinase subunits, M-CK and B-CK subunits, were measured in cultured myogenic cells and in subcultured fibroblasts. In differentiated myogenic cells, the myotubes, both M-CK and B-CK subunits are synthesized. Their rates of degradation were compared. The M-CK subunits is slightly more stable and is degraded with an average apparent half-life of 75 h, whereas that of the B-CK subunit was shorter with 63 h. The turnover properties of M-CK subunit from soluble and of myofibril-bound MM-CK homodimeric creatine kinase isoenzyme isolated from breast muscle of young chickens were identical. The apparent half-life of the B-CK subunit was also determined in subcultured fibroblasts and 5-bromo-2'-deoxyuridine-treated cells, and found to be shorter than in myotubes (46 h and 37 h respectively). Similar observations were made for myosin heavy chain, actin and total acid-precipitable material. It appears therefore that proteins are in general degraded more slowly in differentiated myogenic cells. The differences in the stability of M-CK and B-CK subunits in myotubes probably do not reflect a major regulatory mechanism of the creatine kinase isoenzyme transition.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

The turnover of protein in discrete areas of rat brain

1. Rats were injected serially with [(14)C]glucose to obtain a constant specific radioactivity of brain amino acids. Measurements with this system for periods of up to 8h gave an apparent mean half-life for protein in whole brain of 85h (indicating the presence of a protein fraction with much more rapid turnover than this). 2. The half-lives of proteins in the granule-cell, molecular and white-matter layers of cerebellum were also determined. These had values of 33, 59 and 136h respectively. In addition, the incorporation into protein in six layers of the cerebral cortex, subjacent white matter and five layers of Ammon's horn was studied. All cell-body layers incorporated amino acids at about the same rate irrespective of location, and these rates were considerably higher than those for incorporation into proteins in areas rich in dendrites or fibre tracts. 3. A new method for measuring small amounts of glutamate with a cyclic enzyme system is presented.     

Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts

Puromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues.     

Dynamics of four rat liver plasma membrane proteins and polymeric IgA receptor. Rates of synthesis and selective loss into the bile.

We have determined the half-lives and amounts per hepatocyte of the polymeric IgA receptor (pIgA-R) and four rat hepatocyte plasma membrane proteins and subsequently have predicted their rates of synthesis and possible routes of degradation. Using in vivo pulse-chase metabolic labeling with L-[35S]cysteine, we found that the pIgA-R had an apparent half-life of 1.1 h. Additional metabolic labeling experiments showed that CE9, HA4, and HA321 had apparent half-lives of 4-5 days, and dipeptidyl peptidase IV had an apparent half-life of 9 days. To quantify the amount of each protein per hepatocyte, homogenates and a standard curve of purified protein were compared by immunoblotting. We found that these proteins were present at 1-8 x 10(6) molecules/hepatocyte. The calculated rate of synthesis for pIgA-R was 1.6 x 10(6) molecules/hepatocyte/h, whereas the others were synthesized at much lower rates (0.9-5 x 10(4) molecules/hepatocyte/h). Using immunoblot analysis, we found that pIgA-R was released into bile at a rate of 30%/h (700%/day), whereas dipeptidyl peptidase IV and HA4 were released at a rate of 2-3%/day. While the majority of the loss of pIgA-R from hepatocytes occurred by release into the bile, less than 30% of the degradation of dipeptidyl peptidase IV and HA4 could be accounted for by this pathway, suggesting that the remaining molecules must be retrieved from the apical surface before degradation.     

Degradation of proteins microinjected into cultured mammalian cells.

Iodinated proteins were degraded after injection into HeLa cells at first-order rates with half-lives varying from three hours for the trout monhistone chromosomal protein, HMG-T, -to 60 hours for whale myoglobin. Fluoresceinated-bovine serum albumin (fl-BSA) was degraded almost twice as fast as unmodified BSA. The rate of degradation of 125I-BSA was very similar in eight cell lines of mouse, human, monkey and rat origin. Microinjected proteins were analyzed on SDS-acrylamide gels after injection, and for BSA and immunoglobin G, all remaining intracellular 125I migrated at the molecular weight of the injected proteins. By contrasting, more than 80% of the extracellular 125I chromatographed as iodotyrosine. With the exception of fl-BSA, which exhibited perinuclear accumulation in approximately one-half of the injected cells, autoradiography showed that throughout the period of study the injected proteins remained dispersed in the cytoplasm.