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Ubiquitin conjugation during endoplasmic-reticulum-associated degradation (ERAD) depends on the activity of Ubc7. Here we show that Ubc1 acts as a further ubiquitin-conjugating enzyme in this pathway. Absence of both enzymes results in marked stabilization of an ERAD substrate and induction of the unfolded-protein response (UPR). Furthermore, basic ERAD activity is sufficient to eliminate unfolded proteins under normal conditions. However, when stress is applied, the UPR is required to increase ERAD activity. We thus demonstrate, for the first time, a regulatory loop between ERAD and the UPR, which is essential for normal growth of yeast cells.

A modeling approach termed ‘nicotine bridging’ is presented to estimate exposure to mainstream smoke constituents. The method is based on: (1) determination of harmful and potentially harmful constituents (HPHC) and in vitro toxicity parameter-to-nicotine regressions obtained using multiple machine-smoking protocols, (2) nicotine uptake distributions determined from 24-h excretion of nicotine metabolites in a clinical study, and (3) modeled HPHC uptake distributions using steps 1 and 2. An example of ‘nicotine bridging’ is provided, using a subset of the data reported in Part 2 of this supplement (Zenzen et al., 2012) for two conventional lit-end cigarettes (CC) and the Electrically Heated Cigarette Smoking System (EHCSS) series-K6 cigarette. The bridging method provides justified extrapolations of HPHC exposure distributions that cannot be obtained for smoke constituents due to the lack of specific biomarkers of exposure to cigarette smoke constituents in clinical evaluations. Using this modeling approach, exposure reduction is evident when the HPHC exposure distribution curves between the MRTP and the CC users are substantially separated with little or no overlap between the distribution curves.

The conserved Target Of Rapamycin (TOR) growth control signaling pathway is a major regulator of genes required for protein synthesis. The ubiquitous toxic metalloid arsenic, as well as mercury and nickel, are shown here to efficiently inhibit the rapamycin-sensitive TORC1 (TOR complex 1) protein kinase. This rapid inhibition of the TORC1 kinase is demonstrated in vivo by the dephosphorylation and inactivation of its downstream effector, the yeast S6 kinase homolog Sch9. Arsenic, mercury, and nickel cause reduction of transcription of ribosome biogenesis genes, which are under the control of Sfp1, a TORC1-regulated transcriptional activator. We report that arsenic stress deactivates Sfp1 as it becomes dephosphorylated, dissociates from chromatin, and exits the nucleus. Curiously, whereas loss of SFP1 function leads to increased arsenic resistance, absence of TOR1 or SCH9 has the opposite effect suggesting that TORC1 has a role beyond down-regulation of Sfp1. Indeed, we show that arsenic activates the transcription factors Msn2 and Msn4 both of which are targets of TORC1 and protein kinase A (PKA). In contrast to TORC1, PKA activity is not repressed during acute arsenic stress. A normal level of PKA activity might serve to dampen the stress response since hyperactive Msn2 will decrease arsenic tolerance. Thus arsenic toxicity in yeast might be determined by the balance between chronic activation of general stress factors in combination with lowered TORC1 kinase activity.

Rats were given intravenous injections of l-[1-14C]leucine. Twelve minutes after injection, testes, kidneys, livers, and hepatomas were excised rapidly from one group of animals bearing Morris hepatoma 5123tc. From a second group of rats, the blood was removed 90 min after injection. Tissue extracts and serum were divided into three portions each, and albumin was isolated from each of the three portions by one of three different methods. The three different isolation procedures were the following: (A) pretreatment of the tissue extracts and serum with bovine serum albumin and its specific antiserum and subsequent immunoprecipitation of the rat serum albumin, (B) direct immunoprecipitation followed by dissolving the precipitated rat serum albumin in acid/ethanol, precipitation with ether, and ion-exchange chromatography of the redissolved albumin on CM-cellulose, and (C) a modification of a procedure published previously including fractionation with trichloroacetic acid, ethanol, ether, and ammonium sulfate, chromatography on Sephadex G-100 and DEAE-cellulose, and preparative disc electrophoresis in polyacrylamide gel at pH 10.3 and pH 2.7. With method (A), radiochemically pure albumin can be obtained only from serum. Even though testis and kidney do not synthesize albumin, albumin preparations isolated by this procedure from these organs contain significant amounts of radioactivity. Specific radioactivities measured in albumin prepared by method (A) from the four tissues examined are 5–19 times larger than those in preparations isolated by method (C). Thus, method (A) is inappropriate for the isolation of radiochemically pure albumin from the tissues studied. Procedure (B) is sufficient to obtain radiochemically pure albumin from the serum as well as from the other tissues examined except liver. With liver, this method yields an albumin preparation containing 53% more radioactivity than does albumin isolated with method (C). Method (C) is the only procedure yielding radiochemically pure albumin from all sources, including liver. In liver and hepatoma, the properties of the radioactive impurities are very similar to the properties of albumin itself. Therefore, several purification steps and a careful analysis of the distribution of radioactivity among the albumin fractions after chromatographies and electrophoreses are necessary to separate radioactive impurities from the albumin from homogenates of these two sources.

Intraperitoneally injected radioactive amino acids were efficiently incorporated into the protein of Morris hepatomas 5123tc and 9121 implanted into the muscles of the hind legs of totally hepatectomized rats. However, no radioactive protein was released from the hepatomas into the bloodstream. Albumin synthesis was studied in liver and hepatomas by measuring the incorporation of l -[1-14C]leucine. In these experiments, liver and hepatomas were removed within the time required for synthesis and secretion of albumin. Compared with liver a considerably smaller proportion of total protein radioactivity was found in albumin of the hepatomas, suggesting a reduced albumin synthesis in the hepatomas. Despite its reduced synthesis the content of albumin was much larger in the extravascular compartment of the hepatoma tissue than in liver. No albumin synthesis could be demonstrated in a cell-free protein synthesizing system consisting of liver microsomes, pH 5-fraction and an ATP generating system, provided that albumin was purified to constant specific radioactivity. A relatively complicated purification procedure was found to be required for the isolation of radiochemically pure albumin from liver and hepatoma from animals injected with l -[1-14C]leucine. Immunoprecipitation was found to be adequate for isolation of radiochemically pure albumin only from serum, but not from liver or hepatoma. The reason was the presence of a very highly labeled albumin-like protein in liver and hepatoma. This albumin-like protein was isolated from liver microsomes and characterized. It had the same molecular weight and amino acid composition as albumin, within the limits of variation of the methods, and could be precipitated quantitatively by anti-albumin. The amino acid sequence of the N-terminus of the albumin-like protein differed from that of albumin by an oligopeptide extension consisting of Gly-Val-Phe-Ser-Arg. Injected l -[1-14C]leucine was incorporated first into the albumin-like protein and appeared thereafter in albumin. Quantitative conversion of the albumin-like protein into albumin was observed during biosynthesis of albumin in liver cell suspensions. After initial incubation of the liver cells with l -[1-14C]leucine, followed by further incubation with excess non-radioactive leucine, radioactivity remained constant in total protein, but increased in albumin to about the same extent as it decreased in the albumin-like protein. The evidence indicates that the albumin-like protein is a precursor protein involved in the biosynthesis of albumin. The precursor is converted into albumin by removal of the N-terminal oligopeptide extension. This extension could have a function in the mechanism and the regulation of albumin synthesis and secretion. A model describing such a function is discussed.

Radioactive leucine was injected into the portal vein of rats followed after 15 seconds by a 180 fold excess of nonradioactive leucine. An albumin-like protein in the liver became highly labelled within 15 minutes after injection. After 150 minutes, the radioactivity in the albumin-like protein had decreased to one tenth. In the serum, radioactively labelled albumin started to appear after 15 minutes and increased there-after up to 150 minutes after injection. Radioactivity in albumin within the liver remained constant at a low level. These results suggest that the albumin-like protein is a biological precursor protein of serum albumin, i.e. a proalbumin.

Immunologically isolated albumin from rat liver microsomes separates on DEAE-cellulose into almost equal proportions of an albumin-like protein and authentic albumin. Besides this similarity in immunological properties, both albumin species have almost the same molecular weight and amino acid composition. Furthermore, the amino acid sequences appear to be identical apart from an additional pentapeptide at the N-terminus of the albumin-like protein. It is suggested that the albumin-like protein represents a precursor which is converted to albumin by release of a pentapeptide from the N-terminus.

The mechanism of the biosynthesis of albumin was studied in cell suspensions from rat liver. The cells were prepared by continuous perfusion of the liver in situ with 0.05% collagenase and 0.10% hyaluronidase and incubated under conditions optilnized for the incorporation of amino acids into protein. Seven minutes after starting the incubation l-[1-14C]leucine was added, followed after 25 min by a 15 or 30-min chase with an 830-fold excess of non-radioactive l-leucine. Total protein, an albumin-like protein, and albumin were isolated from samples withdrawn immediately after addition of non-radioactive leucine and after the 15 and 30-min chases. The specific radioactivity of total protein was found to remain constant after addition of the non-radioactive l-leucine, whereas that of the albumin-like protein decreased and that of albumin increased with incubation time. The increase in albumin radioactivity accounted for the decrease in radioactivity of the albuminlike protein, suggesting that the latter is a precursor of albumin. The precursor protein differed from albumin by an oligopeptide extension at the N-terminal end.

Invivo, Morris hepatomas 5123tc and 9121 do not secrete serum proteins into the bloodstream. However, they synthesize albumin, at a reduced rate, if compared with liver. The produced albumin accumulates, leading to an enlargement of the Golgi apparatus and the appearance of intracellular vesicles. The proportion and the absolute amount of albumin found within the hepatoma cells is increased compared with liver.

The sequence of radioactively labelled amino acids at the N-terminus of proalbumin was determined by automated Edman-degradation. [3H] Valine, [3H] phenylalanine or [14Carginine was incorporated into protein in vivo for a time period of 10 min after injection. Since albumin remains unlabelled during this time period (Urban et al., 1976), separation of proalbumin and albumin was not required for this work. Hence, compared to previous methods, a shorter purification procedure could be used which increased the yield of anti-albumin-precipitable protein and reduced the risk of proteolysis. Microsomes were prepared from livers removed 10 min after injection of the radioactively labelled amino acids. A buffer extract of the acetone-dried powder from these microsomes was chromatographed on DEAE-cellulose. All protein obtained after chromatography which could be precipitated with antiserum to serum albumin was isolated by immunoprecipitation and subsequent separation of the antigen-antibody complex. The sequence of radioactive amino acids in this antigen preparation suggests that about 20–25% of proalbumin possessed at the N-terminus the pentapeptide sequence X-Val-Phe-Arg-Arg- whereas 75–80% contained the hexapeptide sequence Arg-X-Val-Phe-Arg-Arg-.

The regulation of albumin synthesis and serum protein secretion was studied in regenerating rat liver by measuring incorporation of 14C-leucine. Albumin was isolated to radiochemical purity, utilizing a method which eliminates the influence of precursor pool changes on protein labeling. Changes in the size of the product pool were measured. The following results were obtained. 1. The time period between intracaval injection of 14Cleucine and the appearance of radioactive protein in the blood ("secretion time") decreased from 15 min for normal animals to a minimum of 10 min at 48 hours after removal of 70% of the liver. 2. At 10 min after intracaval injection of 14C-leucine, 3.5% of total protein radioactivity was found in albumin in normal liver, whereas in regenerating liver only 1.4% of the total protein radioactivity was incorporated into albumin. 3. Albumin concentration in the serum decreased from 29.2 mg of albumin per ml of serum for normal rats to a minimum of 17.3 mg of albumin per ml of serum at 4 days after partial hepatectomy. 4. The half-life of albumin was 2.66 days for normal rats and 2.13 days for animals, 48 hours after partial hepatectomy. 5. The intravascular pool of albumin decreased from 100 mg of albumin per 100 g, body wt, in normal rats to 57.8 mg of albumin per 100 g, body wt, in partially hepatectomized animals, 48 hours after operation. The extravascular and the total body pool of albumin also decreased after partial hepatectomy to a minimum at 24 hours after the operation. In contrast to the intravascular pool, the extravascular and the total body pool increased again, reaching a plateau between 2 and 4 days after the operation. 6. During regeneration, the proportion of leucine to other amino acids in total liver protein did not change. Also, this proportion did not differ significantly from that found in serum albumin. 7. The net rate of albumin synthesis changed only slightly from 20.1 mg of albumin per day per g of liver for normal to 23.9 mg albumin per day per g of liver for regenerating liver 48 hours after partial hepatectomy. In contrast, the net rate of synthesis of total liver protein increased from 576 mg protein per day per g of liver in normal rats to 1710 mg of protein per day per g of liver in rats 48 hours after partial hepatectomy.