Smooth Endoplasmic Reticulum Fractions Research Articles

Enrichment of microsomes from Chinese hamster ovary cells by subcellular fractionation for its use in proteomic analysis.

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30–60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10–60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10–60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.

Retention of a co-translational translocated mutant protein of carboxypeptidase Y of Saccharomyces cerevisiae in endoplasmic reticulum.

Co-translational translocation of Saccharomyces cerevisiae vacuolar glycoprotein carboxypeptidase Y (CpY) was highly efficient when studied with an in vivo and in vitro homologous system, comparison of limited proteolytic cleavage of immunoprecipitated translational products of CpY and subcellular localisation of a mutant CpY. The efficient segregation of CpY mRNA in highly purified fractions of rough microsomes was characterised. CpY1 mutant showed retention of core glycosylated material (proCpY1) in the rough and smooth endoplasmic reticulum fractions. It is suggested that the presence of structures that are incompatible with intercompartmental transport of vacuolar protein leads to retention of the mutated CpY by the endoplasmic reticulum.

Subcellular localization of non-specific carboxylesterases, acylcarnitine hydrolase, monoacylglycerol lipase and palmitoyl-CoA hydrolase in rat liver

The subcellular distribution and sidedness on the membranes of four chemically and genetically distinct esterases (esteraces ES-3, ES-4, ES-8, ES-15) in rat liver was investigated using selective substrates. (1) Rat liver homogenate was divided into nine subcellular fractions by differential centrifugation techniques. The cell fractions were assayed for the enzymatic hydrolysis of acetanilide (ES-3), propanidid, palmitoyl-CoA and monopalmitolglycerol (ES-4), methyl butyrate and octanolglyceryol (ES-8), and decanoylcarnitine (ES-15). With all substrates, the highest specific activities were found in the rough and smooth endoplasmic reticulum fractions. This localization of the esterases was confirmed by labelling the cell fractions with the specific, covalently binding inhibitor (bis(4-nitro[ 14C]phenyl) phosphate. The enzymatic hydrolysis of the palmitoyl esters in differing cell fractions did not completely parallel that of propanidid. This confirms the well-known existence of palmitoyl-CoA hydrolases other than esterase ES-4. (2) Density gradient fractions with crude mitochondria indicated that a low amount of at least one of these carboxylesterases was an integral part of these organelles too. (3) Proteinase treatment reduced the non-specific esterase activities as well as lipase activities versus dioctanolglycerol, acylcarnitines and palmityl-CoA only in detergent-disrupted microsomal vesicles. This might indicate a lumenal orientation of these enzymes. However, of the charged substrates palmitoylcarnitine and palmitoyl-CoA only the latter one showed the typical latency to be expected for a hydrolysis in the lumen of the endoplasmic reticulum.

Hepatic Ultrastructure and Biochemistry Following Insulin Withdrawal

This study examines the hepatic ultrastructural and biochemical responses following insulin withdrawal from insulin-maintained streptozotocin- diabetic rats. This hormonal manipulation permitted analysis of the hepatocellular events occurring during diabetic onset. Normoglycemia was maintained in the induced diabetic rats for seven days with daily injections of 5 U intermediate acting insulin (NPH-Iletin) per 100 gram body weight. This was followed by treatment for 24 hr with regular short acting insulin at 6 hr intervals (2.5 U/100 gr b.w.).Animals were sacrificed at intervals following the last insulin injection. Plasma was collected for glucose and insulin determinations. Liver samples were prepared for ultrastructural analysis or were collected for Isolation of rough and smooth endoplasmic reticulum fractions (RER, SER). Isolated RER and SER membrane preparations were analyzed for glucose-6-phosphatase (G-6-Pase) activity, as well as for phospholipid, cholesterol, and fatty acid composition.

Association of poliovirus proteins with the endoplasmic reticulum.

Poliovirus proteins, except P3-7c, are associated with the endoplasmic reticulum after extraction of the cytoplasm and centrifugation of membranes to equilibrium in sucrose gradients. Proteins P3-2, P2-X, and P3-9 are found preferentially among the rough endoplasmic reticulum, whereas P3-7c is located in smooth endoplasmic reticulum fractions. P3-7c is probably not membrane associated, since it can be separated from membranes after centrifugation in buffer. However, P3-4a, P2-5b, P2-X, and P3-9 are avidly bound to membranes and cannot be dislodged with high-ionic-strength buffer containing EDTA or 4 M urea. These proteins are digested by trypsin, indicating peripheral rather than internal localization.

Effects of luteinizing hormone withdrawal on Leydig cell smooth endoplasmic reticulum and steroidogenic reactions which convert pregnenolone to testosterone.

Depletion of endogenous LH with sc implants of testosterone-17 beta-estradiol (T-E) caused a reduction in the Leydig cell smooth endoplasmic reticulum (SER) over a 10-day treatment period. Decreases also occurred in some, but not all, of the testicular steroidogenic reactions responsible for the conversion of pregnenolone (PREG) to testosterone. The conversions of progesterone (PROG) to 17 alpha-hydroxyprogesterone, 17 alpha-hydroxyprogesterone to androstenedione, and androstenedione to testosterone were significantly correlated (P less than 0.05) with the loss of Leydig cell SER. In contrast, the testicular conversion of PREG to PROG in rats deprived of endogenous LH for up to 10 days was identical to that in intact controls. Similar results were obtained when rats were hypophysectomized for 10 days. These results indicate that the Leydig cell enzyme activities responsible for converting PREG to PROG are distributed in the Leydig cell SER fraction which remains in Leydig cell cytoplasm 10 days after LH withdrawal, and thus, the bulk of these enzyme activities are sequested in a SER compartment that is resistant to LH withdrawal.

Heterogeneity of cyclic nucleotide phosphodiesterases in liver endoplasmic reticulum.

A microsomal fraction from rat liver was subfractionated into three rough endoplasmic reticulum fractions RIII, RII and RI, together with a smooth endoplasmic reticulum plus Golgi fraction. Cyclic nucleotide phosphodiesterase activity was found in all fractions. Subsequently it was shown that Golgi fractions were essentially devoid of cyclic AMP phosphodiesterase activity and the activity resided in the smooth endoplasmic reticulum fraction. The activity of the endoplasmic reticulum constituted some 20% of the homogenate activity, with the major fraction of this being associated with the RII fraction and the least with the RI fraction. With the exception of the activity of the RI fraction, which was a peripheral enzyme, all of the other enzyme activities were integral, requiring detergent or repeated freeze-thawing to effect solubilization. All of the activities appeared to be exposed at the external surface of the endoplasmic reticulum, as they were inactivated by trypsin under conditions where glucose 6-phosphatase was not. All of these activities displayed distinct sensitivities to both thermal and trypsin inactivation, yielding activity decays consistent with a single enzyme species being present in each case. The freeze-thaw-solubilized enzymes yielded single symmetrical peaks on sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The sedimentation coefficients for the enzymes in the smooth-endoplasmic-reticulum-plus-Golgi, RIII, RII and RI fractions were 3.2S, 4.2S, 4.5S and 4.5S respectively. Whereas the activity in the smooth-endoplasmic-reticulum-plus-Golgi fraction exhibited normal Michaelis kinetics, those in the other fractions yielded kinetics indicative of apparent negative co-operativity. All of the enzymes exhibited low Km values towards cyclic AMP. The enzymes did not appear to be regulated by Ca2+ or calmodulin. ZnCl2 was found to be a potent non-competitive inhibitor of the enzyme in all fractions. NaF was a weak non-competitive inhibitor. The bilayer fluidizing agent benzyl alcohol exerted dissimilar effects on the enzyme activities. It is concluded that the endoplasmic reticulum displays lateral heterogeneity, with single, rather distinct, cyclic AMP phosphodiesterases being found in the different fractions.

The submicrosomal distribution of dolichol and dolichol phosphokinase activity in rat liver.

Microsomes were isolated from rat liver and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER) components, and the purity of these fractions was determined. The dolichol contents of each of the three fractions was estimated, using high-pressure liquid chromatography. Although highest concentrations (1940 ng/mg protein) were found in Golgi, the RER contained the largest absolute amounts. The presence of large quantities of dolichol in RER is consistent with the role of dolichol as an intermediate in asparagine-linked glycoprotein synthesis. RER and SER fractions contained high specific activities for dolichol phosphokinase, while the activity in Golgi was quite low. High concentrations of dolichol in Golgi and high dolichol phosphokinase activity in SER suggest that dolichol (and dolichyl phosphate) may be utilized in Golgi for glycoprotein processing and in the transmembrane movement of sugars such as galactose.

Transport of newly synthesized vesicular stomatitis viral glycoprotein to purified Golgi membranes.

In a previous communication we reported that the newly synthesized membrane glycoprotein of vesicular stomatitis virus could be transported in crude extracts of CHO cells from endoplasmic reticulum-derived membranes to membranes of the Golgi complex. This conclusion was an indirect one, based on the terminal glycosylation of this glycoprotein, a reaction that was dependent upon a Golgi-specific enzyme, UDP-GlcNAc transferase I. We show here that the Golgi fraction of rat liver will substitute for members of CHO cells as a source of transferase I in this reaction. The use of highly purified fractions of liver Golgi membranes, coupled with the ability to recover these membranes from incubations, has now permitted a direct demonstration of net transport of G protein to these heterologous Golgi membranes. This transport reaction is specific, in that the smooth endoplasmic reticulum fraction will not substitute for the Golgi fraction, is quantitatively significant, involving at least 30% of the viral glycoprotein, and is sustained only in the presence of both ATP and a soluble, cytosol fraction of liver cells.

Effects of repeated administration of rat 2-diethylaminoethyl-2-2-diphenylvalerate-HCl (SKF 525 A) on liver

Repetitive administration to male rats of 2-diethylaminoethyl-2-2-diphehylvalerate-HCl (SKF 525 A) (50 mg/kg i.p.), decreases the intensity of [ 14C]-orotic acid incorporation/mg of RNA but not the 14C-incorporation/g liver. The RNA content/g liver is significantly higher in SKF-treated animals than in controls. Decay of label in liver RNA from [ 14C]orotic acid pretreated animals, is not significantly different in SKF 525 A treated animals than in controls. SKF 525 A repetitive administration, does not modify the rate of incorporation of 32P in liver microsomal lipid when results are expressed per μg of inorganic phosphorus but it does when expressed in terms of per gram liver. There is a significant decrease in the decay rate of label from 32P-prelabeled liver microsomal phospholipids when animals are treated with SKF 525 A. There is a significant increase in the protein and phospholipid content in the smooth endoplasmic reticulum fraction. The electron microscopy of liver from SKF 525 A-treated animals, shows the presence of large areas of round vesicles of swollen endoplasmic reticulum, partly due to smooth component and part due to rough component, having detached the ribosomes from their membranes. Results suggest an inhibitory effect of SKF 525 A on RNA and phospholipid degradative processes.

In vivo effect of colchicine on hepatic protein synthesis and on the conversion of proalbumin to serum albumin.

Treatment of rats with 0.5-25 mumol/100 g body weight of colchicine for 1 h or more caused an inhibition of hepatic protein synthesis. This effect was not seen if animals were exposed to colchicine for less than 1 h. The delayed inhibition of protein synthesis affected both secretory and nonsecretory proteins. Treatment with colchicine (15 mumol/100 g) for 1 h or more caused the RNA content of membrane-bound polysomes to fall but did not change the polysomal profile of this fraction. By contrast, the total RNA content in the free polysome cell fraction was increased, and this was due to the presence of more ribosomal monomers and dimers. Electron microscope examination of the livers from rats treated for 3 h with colchicine showed an accumulation of secretory vesicles within the hepatocytes and a general distention of the endoplasmic reticulum. Administration of radioactive L-leucine to the rats led to an incorporation of radioactivity into two forms of intracellular albumin which were precipitable with antiserum to rat serum albumin but which were separable by diethylaminoethyl-cellulose chromatography. One form has arginine at the amino-terminal position and is proalbumin, and the other form, which more closely resembles serum albumin chromatographically, has glutamic acid at its amino terminus. Only proalbumin was found in rough and smooth endoplasmic reticulum fractions and in a Golgi cell fraction wich corresponds morphologically to mostly empty and partially filled secretory vesicles. However, in other Golgi cell fractions which were filled with secretory products, both radioactive proalbumin and serum albumin were found. This indicates that proalbumin is converted to serum albumin in these secretory vesicles just before exocytosis. Colchicine delayed the discharge of radioactive albumin from these filled secretory vesicles and caused an accumulation of both proalbumin and serum albumin within these cell fractions.

Specific binding of prostaglandins E2 and F2alpha by membrane preparations from rat skin.

Specific binding of [3H]prostaglandin E2 ([3H]PGE2) and [3H]prostaglandin F2 alpha ([3H]PGF2 alpha) to plasma membrane and smooth endoplasmic reticulum fractions prepared from rat skin was demonstrated by the Millipore filter assay system. Specific binding was greater in the smooth endoplasmic reticulum fraction. Maximum binding was attained in the presence of Ca++ (0.5 x 10(-3) M), pH 7.2, and at a temperature of 37 C. Unlabeled PGE2 at a concentration of approximately 700 x 10(-9) M inhibited binding to the smooth endoplasmic reticulum fraction by approximately 90%. Other unlabeled prostaglandins, PGE1, PGF2 alpha, and a PGE2 analog, 7-0-13-prostynoic acid, at the same concentration inhibited binding by 40%, 25%, and 30%, respectively. A variety of unlabeled fatty acids (palmitic, oleic, linoleic, eicosatrienoic, and eicosatetraenoic acids), at the higher concentration of approximately 1700 x 10(-9) M, inhibited binding less than 30%, suggesting the presence of specific receptors for PGE2 in the smooth endoplasmic reticulum. Similar results were obtained for the competitive binding studies with [3H]PGF2 alpha. Proteolytic digestion of the membrane fraction by trypsin and pronase, or boiling for 15 min caused marked inhibition of binding, suggesting that the receptors for PGE2 and PGF2 alpha have a protein component. The Scatchard plot analysis of the equilibrium-binding data of [3H]PGE2 and [3H]PGF2 alpha to normal smooth endoplasmic reticulum fractions revealed an apparent dissociation constant (Kd) of 1.1 x 10(-9) M with a binding site concentration of 75 x 10(-12) M for [3H]PGE2, and a Kd of 1,0 x 10(-9) M with a binding site concentration of 35 x 10(-12) M for [3H]PGF2 alpha. These data indicate a greater concentration of binding sites for PGE2 than PGF2 alpha in the normal skin smooth endoplasmic reticulum. On the other hand, analysis of smooth endoplasmic reticulum from skin of essential fatty acid-deficient rats revealed a Kd of 1.2 x 10(-9) M with a binding site concentration of 75 x 10(-12) M for [3H]PGE2, and a Kd of 2.2 x 10(-9) M with a binding site concentration of 175 x 10(-12) M for [3H]PGF2 alpha. The concentration of binding sites for PGF2 alpha in the smooth endoplasmic reticulum of the skin of these rats is increased 5-fold when compared to normal values, whereas that of PGE2 was not altered. These results suggest a possible alteration of PGF2 alpha specific binding to skin endoplasmic reticulum during the pathophysiological abnormalities that accompany essential fatty acid-deficiency syndrome.

Studies on the site of addition of sialic acid and glucosamine to rat alpha 1-acid glycoprotein.

Ultrasonic extracts of rough and smooth endoplasmic reticulum fraction and Golgi fractions from rat liver were examined by immunoelectrophoresis using antiserum to alpha 1-acid glycoprotein. Rough endoplasmic reticulum fractions contained only sialic acid free alpha 1-acid glycoprotein, whereas smooth endoplasmic reticulum and Golgi fractions also contained sialic acid containing alpha 1-acid glycoprotein. Determination of the sialic acid contents of immune precipitates isolated from the extracts suggested that the Golgi complex was the main site of addition of sialic acid to alpha 1-acid glycoprotein. Immunological studies on puromycin extracts of polyribosomes showed that polypeptide chains of alpha 1-acid glycoprotein and albumin were assemble mainly on membrane-bound polyribosomes. Evidence is presented from incorporation studies with labelled leucine and glucosamine that initial glycosylation of alpha 1-acid glycoprotein occurs mainly or entirely after release of nascent polypeptide from the ribosomal site.

Calcium-stimulated adenosine triphosphatase in the microsomal fraction of tooth germ from porcine fetus

The characterization and localization of a Ca 2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in the tooth germ of the porcine fetus are reported. This enzyme, a microsome fraction, is preferentially activated by Ca 2+. In the presence of 0.5 mM ATP, maximal enzyme activity is obtained at 0.5–1.0 mM CaCl 2. The maximal rate of ATP hydrolysis is approx. 20 μmol per h per mg of protein as the enzyme preparation is used here. At optimal Ca 2+ concentration, the Mg 2+ has an inhibitory effect. The enzyme does not require Na + or/and K + for activation by Ca 2+. Other nucleotide triphosphates may serve as the substrate, but V for ATP is the highest. The K m for ATP is 8.85 · 10 −5 M. The optimal pH for Ca 2+ activation of the enzyme lies around 9.2. Well known inhibitors of (Na + + K +)-ATPase, mitochondria ATPase and Ca 2+-ATPase in the erythrocyte do not inhibit the enzyme. In the subcellular order the enzyme may be assumed to be localized in the smooth endoplasmic reticulum fraction containing cell and Golgi body membrane fragments and in the tissue order in the enamel organ containing an ameloblast layer, stratum intermedium and stellate reticulum.

The induction of aryl hydrocarbon and estrogen hydroxylases in the rough and smooth endoplasmic reticulum in regenerating rat liver

In normal liver the aryl hydrocarbon hydroxylase (AHH) activity is twice as great in the smooth endoplasmic reticulum fraction as in the rough fraction. In the hepatectomized animal, however, the ratio becomes 1 : 1. Benzanthracene treatment brings about a 12-fold induction in the endoplasmic reticulum (ER) fractions of partially hepatectomized livers, and a smaller increase in these fractions from normal livers. In the case of estrogen hydroxylase (EH), the majority of the basal activity is also in the smooth fraction. Hepatectomy again brings about a 1 : 1 ratio, and 1,2-benzanthracene (BA) brings about a 2-fold increase in the activity of both fractions. The inducer, however, has no effect on the activity of this enzyme in either fraction from normal liver. Both activities show a very small increase when benzanthracene is given at the time of partial hepatectomy. Benzanthracene injection between 2 and 8 h after hepatectomy causes an initial increase in the rough endoplasmic reticulum activities followed by an increase in the smooth fraction activities. No further increase in hydroxylase activities in the rough membrane fraction occurs when benzanthracene is injected more than 8 h after hepatectomy but these activities of the smooth membrane fraction now increase. Actinomycin D blocks the induction of AHH in the rough fraction completely, but some of the induced activity remains in the smooth fraction. Puromycin has a similar effect. In the case of EH activity both antibiotics completely stop the induction. It is suggested that different mechanisms control the activity levels of these two microsomal mixed-function oxidase systems.

Phospholipid metabolism in endoplasmic reticular membranes in rats fed high-carbohydrate diet

1. 1. The composition and metabolism of phosphatidylcholine and phosphatidylethanolamine components of the rough and smooth endoplasmic reticulum fractions in liver have been studied in rats fed chow, fasted 48 h and refed with high-carbohydrate diet after 48 h fasting. 2. 2. High-carbohydrate fed rats showed a large increase in neutral lipid levels in liver and plasma. Labelling of [1- 14C]palmitic acid in liver triglycerides was higher in this group of rats. Compared to the chow-fed rats, there was a reduced incorporation of fatty acid into plasma triglycerides of the high-carbohydrate fed rats. 3. 3. Phosphatidylethanolamine levels showed a marked decrease in both smooth and rough endoplasmic reticulum fractions of high-carbohydrate fed rats. Phosphatidylcholine levels showed a significant decrease only in the rough microsome fraction in these rats. 4. 4. Linoleic and arachidonic acid concentrations of the phospholipids decreased to a low level in the submicrosomal fractions of high-carbohydrate fed rats. These two fatty acids were virtually absent (a decrease of 92–96%) in the phospholipids of rough microsome fraction. Fatty acid incorporation into the phospholipids of the rough microsomes was almost completely inhibited in high-carbohydrate fed rats. 5. 5. Alterations of submicrosomal phospholipid metabolism is probably related to the changes in lipogenesis and in lipid transport observed on refeeding high-carbohydrate diet.

The effect of vitamin E on the intracellular distribution of the different oxidation states of selenium in rat liver.

1. The liver intracellular distribution of (75)Se, (75)Se(2-) and (75)SeO(3) (2-) formed from orally administered Na(2) (75)SeO(3) was studied in rats given four different dietary treatments. 2. Subcellular fractionation was done by using sucrose density gradients in a B XIV zonal centrifuge rotor, and conditions were established so that separation of lysosomal, mitochondrial, smooth- and rough-surfaced endoplasmic reticulum, and soluble fractions was achieved. 3. Marker enzymes acid phosphatase, succinate-2 - p - iodophenyl - 3 - p -nitrophenyl - 5 - phenyltetrazolium reductase and glucose 6-phosphatase were used, together with electron microscopy, to establish the identity of the fractions. 4. The dietary treatments investigated were: (a) vitamin E-deficient diet for 3 months, re-fed with vitamin E during the terminal 5 days; (b) vitamin E-deficient diet; (c) adequate diet; (d) vitamin E- and selenium-deficient diet, re-fed with vitamin E during the terminal 5 days. 5. In adequately fed rats, selenide was particularly associated with the mitochondrial fractions; in vitamin E-deficient rats, little selenide was found and the buoyant density of the mitochondria was increased, whereas re-feeding with vitamin E showed a restoration of the normal pattern. In vitamin E- and selenium-deficient rats, re-fed with vitamin E, there was no tendency for selenide to be localized in the mitochondria. 6. In the microsomal regions of the gradients, adequately fed rats showed a concentration of selenide, particularly in the smooth endoplasmic reticulum fractions, and to a lesser extent in the rough endoplasmic reticulum fractions. This was not observed in vitamin E-deficient rats, and the normal pattern was restored on re-feeding with vitamin E, both in rats given the vitamin E-deficient diet and the vitamin E- and selenium-deficient diet. 7. Some selenide was also found in the soluble fractions, when vitamin E was present, and a substantial proportion of this selenide was found to pass through a dialysis membrane. 8. These results are taken to support our hypothesis that the active form of selenium may be selenide located in non-haem iron-containing proteins, and that the function of vitamin E may be to protect the selenide from oxidation.

Size Distribution of Membrane Proteins of Rat Liver and Their Relative Rates of Degradation

Abstract The proteins associated with various membrane fractions, including smooth and rough endoplasmic reticulum, plasma membrane, and mitochondria of rat liver, were solubilized with sodium lauryl sulfate and separated by electrophoresis on sodium lauryl sulfate acrylamide gels. Smooth and rough endoplasmic reticulum have similar electrophoretic patterns. All other fractions have characteristic patterns in which no single or predominant protein occurs. The smooth endoplasmic reticulum fraction was fractionated by use of Triton X-100, which solubilized approximately 50% of the membrane proteins. The patterns of Triton X-100-soluble and -insoluble proteins are distinctive. The relative rates of degradation of the proteins were studied by use of a double isotope technique in which 14C-leucine was administered to intact rats 4 days prior to administration of 3H-leucine. As indicated by the differences in 3H:14C ratios, there is a marked heterogeneity of turnover of proteins of both the endoplasmic reticulum and plasma membrane. There is a general correlation between the relative degradation rates of membrane proteins and the molecular size of the protein (subunit) as separated by electrophoresis in sodium lauryl sulfate acrylamide gels. This general relationship was found also for the soluble protein fraction of rat liver, whether fractionated by sodium lauryl sulfate acrylamide gels, or by Sephadex G-200 columns in the presence of sodium lauryl sulfate. A model in which membrane proteins associated and dissociate from the protein-lipid complex is favored to explain the heterogeneity of degradation, and the general correlation of size with degradation rate.