Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.

The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function.     

Cell-to-cell transport via the lumen of the endoplasmic reticulum

Plasmodesmata are plasma membrane‐lined channels through which cytoplasmic molecules move from cell‐to‐cell in plants. Most plasmodesmata contain a desmotubule, a central tube of endoplasmic reticulum (ER), that connects the ER of adjacent cells. Here we demonstrate that molecules of up to 10.4 kDa in size can move between the ER lumen of neighbouring leaf trichome or epidermal cells via the desmotubule lumen. Fluorescent molecules of up to 10 kDa, microinjected into the ER of Nicotiana trichome cells, consistently moved into the ER and nuclei of neighbouring trichome cells. This movement occurred more rapidly than movement via the cytoplasmic pathway. A fluorescent 3‐kDa dextran microinjected into the ER of a basal trichome cell moved into the ER and nuclei of epidermal cells across a barrier to cytoplasmic movement. We constructed a 10.4‐kDa recombinant ER‐lumenal reporter protein (LRP) from a fragment of the endogenous ER‐lumenal binding protein AtBIP1. Following transient expression of the LRP in the ER of Tradescantia leaf epidermal cells, it often moved into the nuclear envelopes of neighbouring cells. However, green fluorescent protein targeted to the ER lumen (ER‐GFP) did not move from cell to cell. We propose that the ER lumen of plant cells is continuous with that of their neighbours, and allows movement of small ER‐lumenal molecules between cells.     

Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum

The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5′-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphates (r >/ 0.98) and negatively with the plasma membrane marker 5′-nucleotidase (r ranging between ?0.88 and ?0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum.     

Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum.

The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5'-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphatase (r larger than or equal to 0.98) and negatively with the plasma membrane marker 5'-nucleotidase (r ranging between -0.88 and -0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum.     

The endoplasmic reticulum in regenerating liver cells

Summary Light-microscopic analysis of mouse liver homogenates six days after partial hepatectomy, showed a higher percentage of nuclei with adherent cytoplasm than homogenates from normal liver. This observation was true for animals with either a slow or rapid recovery of body weight after the operation. The phenomenon was not a function of the changes in the proportions of parenchymal and non-parenchymal tissue in the regenerating liver.Electron-microscopic analysis of random samples from normal and regenerating livers indicated an increase in the perinuclear rough endoplasmic reticulum, and a displacefment of the glycogen depots within the regenerating cells six days after partial hepatectomy.The marked resistance towards homogenization, shown by the cytoplasm of the regenerating cells, may have been due to the observed increase of perinuclear membranes. However, qualitative changes of the cell membranes and a general decrease of proteolytic activity connected with liver regeneration may also have contributed.     

Localization of the N-terminal methionine of rat liver cytochrome P-450 in the lumen of the endoplasmic reticulum

Recent cumulative evidence suggests that liver microsomal cytochrome P-450 (P-450) is exposed to the cytosol with the exception of the N-terminal peptide (amino acid residues 1 to 21), or two peptides (residues 1 to 60). We tested the localization of the N-terminal methionine residue of P-450IIB1 of rat liver microsomes in the natural membrane with the site-specific reagent fluorescein isothiocyanate. The N-terminus of isolated P-450 was stoichiometrically modified in solution with fluorescein isothiocyanate. In intact microsomes, the N-terminus was not modified but became accessible to the reagent when the membrane was dissolved with Triton X-100. Our results indicate that the N-terminus faces the lumen of the endoplasmic reticulum, and we propose that P-450 spans the membrane only once with amino acid residues 1 to 21.     

Signal sequences initiate the pathway of maturation in the endoplasmic reticulum lumen

An interaction between an N-terminal signal sequence and the translocon leads to the initiation of protein translocation into the endoplasmic reticulum lumen. Subsequently, folding and modification of the substrate rapidly ensue. The close temporal coordination of these processes suggests that they may be structurally and functionally coordinated as well. Here we show that information encoded in the hydrophobic domain of a signal sequence influences the timing and efficiency of at least two steps in maturation, namely N-linked glycosylation and signal sequence cleavage. We demonstrate that these consequences correlate with and likely stem from the nature of the initial association made between the signal sequence and the translocon during the initiation of translocation. We propose a model by which these maturational events are controlled by the signal sequence-translocon interaction. Our work demonstrates that the pathway taken by a nascent chain through post-translational maturation depends on information encoded in its signal sequence.     

Diffusion of green fluorescent protein in the aqueous-phase lumen of endoplasmic reticulum

The endoplasmic reticulum (ER) is the major compartment for the processing and quality control of newly synthesized proteins. Green fluorescent protein (GFP) was used as a noninvasive probe to determine the viscous properties of the aqueous lumen of the ER. GFP was targeted to the ER lumen of CHO cells by transient transfection with cDNA encoding GFP (S65T/F64L mutant) with a C-terminus KDEL retention sequence and upstream prolactin secretory sequence. Repeated laser illumination of a fixed 2-micrometers diameter spot resulted in complete bleaching of ER-associated GFP throughout the cell, indicating a continuous ER lumen. A residual amount (<1%) of GFP-KDEL was perinuclear and noncontiguous with the ER, presumably within a pre- or cis-Golgi compartment involved in KDEL-substrate retention. Quantitative spot photobleaching with a single brief bleach pulse indicated that GFP was fully mobile with a t1/2 for fluorescence recovery of 88 +/- 5 ms (SE; 60x lens) and 143 +/- 8 ms (40x). Fluorescence recovery was abolished by paraformaldehyde except for a small component of reversible photobleaching with t1/2 of 3 ms. For comparison, the t1/2 for photobleaching of GFP in cytoplasm was 14 +/- 2 ms (60x) and 24 +/- 1 ms (40x). Utilizing a mathematical model that accounted for ER reticular geometry, a GFP diffusion coefficient of 0.5-1 x 10(-7) cm2/s was computed, 9-18-fold less than that in water and 3-6-fold less than that in cytoplasm. By frequency-domain microfluorimetry, the GFP rotational correlation time was measured to be 39 +/- 8 ns, approximately 2-fold greater than that in water but comparable to that in the cytoplasm. Fluorescence recovery after photobleaching using a 40x lens was measured (at 23 degrees C unless otherwise indicated) for several potential effectors of ER structure and/or lumen environment: t1/2 values (in ms) were 143 +/- 8 (control), 100 +/- 13 (37 degrees C), 53 +/- 13 (brefeldin A), and 139 +/- 6 (dithiothreitol). These results indicate moderately slowed GFP diffusion in a continuous ER lumen.     

Isocitrate dehydrogenase: A NADPH-generating enzyme in the lumen of the endoplasmic reticulum

The aim of the present study was the investigation of the occurrence of NADPH-generating pathways in the endoplasmic reticulum others then hexose-6-phosphate dehydrogenase. A significant isocitrate and a moderate malate-dependent NADP⁺ reduction were observed in endoplasmic reticulum-derived rat liver microsomes. The isocitrate-dependent activity was very likely attributable to the appearance of the cytosolic isocitrate dehydrogenase isozyme in the lumen. The isocitrate dehydrogenase activity of microsomes was present in the luminal fraction; it showed a strong preference towards NADP⁺versus NAD⁺, and it was almost completely latent. Antibodies against the cytosolic isoform of isocitrate dehydrogenase immunorevealed a microsomal protein of identical molecular weight; the microsomal enzyme showed similar kinetic parameters and oxalomalate inhibition as the cytosolic one. Measurable luminal isocitrate dehydrogenase activity was also present in microsomes from rat epididymal fat. The results suggest that isocitrate dehydrogenase is an important NADPH-generating enzyme in the endoplasmic reticulum.     

The interaction of caldesmon with the COOH terminus of actin

Caldesmon interacts with the NH2-terminal region of actin. It is now shown in airfuge centrifugation experiments that modification of the penultimate cysteine residue of actin significantly weakens its binding to caldesmon both in the presence and absence of tropomyosin. Furthermore, as revealed by fluorescence measurements, caldesmon increases the exposure of the COOH-terminal region of actin to the solvent. This effect of caldesmon, like its inhibitory effect on actomyosin ATPase activity, is enhanced in the presence of tropomyosin. Proteolytic removal of the last three COOH-terminal residues of actin, containing the modified cysteine residue, restores the normal binding between caldesmon and actin. These results establish a correlation between the binding of caldesmon to actin and the conformation of the COOH-terminal region of actin and suggest an indirect rather than direct interaction between caldesmon and this part of actin.     

Effects of lipid peroxidation on membrane-bound enzymes of the endoplasmic reticulum

1. Induction of the formation of lipid peroxide in suspensions of liver microsomal preparations by incubation with ascorbate or NADPH, or by treatment with ionizing radiation, leads to a marked decrease of the activity of glucose 6-phosphatase. 2. The effect of peroxidation can be imitated by treating microsomal suspensions with detergents such as deoxycholate or with phospholipases. 3. The substrate, glucose 6-phosphate, protects the glucose 6-phosphatase activity of microsomal preparations against peroxidation or detergents. 4. The loss of glucose 6-phosphatase activity is not due to the formation of hydroperoxide or formation of malonaldehyde or other breakdown products of peroxidation, all of which are not toxic to the enzyme. 5. All experiments lead to the conclusion that the loss of activity of glucose 6-phosphatase resulting from peroxidation is a consequence of loss of membrane structure essential for the activity of the enzyme. 6. In addition to glucose 6-phosphatase, oxidative demethylation of aminopyrine or p-chloro-N-methylaniline, hydroxylation of aniline, NADPH oxidation and menadione-dependent NADPH oxidation are also strongly inhibited by peroxidation. However, another group of enzymes separated with the microsomal fraction, including NAD⁺/NADP⁺ glycohydrolase, adenosine triphosphatase, esterase and NADH–cytochrome c reductase are not inactivated by peroxidation. This group is not readily inactivated by treatment with detergents. 7. Lipid peroxidation, by controlling membrane integrity, may exert a regulating effect on the oxidative metabolism and carbohydrate metabolism of the endoplasmic reticulum in vivo.     

The transfer of mannose to dolichol diphosphate oligosaccharides in pig liver endoplasmic reticulum

The transfer, catalysed by pig liver microsomal preparations, of mannose, from GDP-mannose, to lipid-linked oligosaccharides and the properties of the products are described. Solubility, hydrolytic and chromatographic data suggest that they are dolichol diphosphate derivatives. The presence of two N-acetyl groups in at least part of the heterogenous oligosaccharide portion was tentatively deduced. Reduction with borohydride of the oligosaccharide showed that the newly added mannose residues were not at its reducing end. Periodate oxidation suggested that 60% of these were at the non-reducing terminus and that 40% were positioned internally. T.l.c. showed the presence of seven oligosaccharide fractions with chromatographic mobilities corresponding to glucose oligomers with 7-13 residues. The molar proportions of the oligosaccharide fractions in the mixture were determined by borotritiide reduction and the number of mannose residues added to each oligosaccharide fraction during the incubation was calculated. Two of the oligosaccharide fractions had received on average one, or slightly more than one, mannose residue per chain during the incubation; four of the other fractions were each shown to be a mixture, 20-25% of which had received one mannose residue during the incubation and 75-80% of which had not been mannosylated during the incubation. This supported other evidence for the presence of endogenous lipid-linked oligosaccharides in the microsomal preparation which had been formed before the incubation in vitro. Evidence for the possibility of two pools of dolichol monophosphate mannose, one being more closely associated with mannosyl transfer to dolichol diphosphate oligosaccharides than the other, is also discussed.     

Processing of human cytomegalovirus UL37 mutant glycoproteins in the endoplasmic reticulum lumen prior to mitochondrial importation

The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues -1 and -3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic alpha-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37(M), a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH(2)-terminal fragment, pUL37(M-NH2), was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37(M-COOH), was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH(2)-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37(M), and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.     

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune‐dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species‐dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA‐dependent secretory pathway. This led to a general inhibition of protein secretion by PDT‐treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin‐based PDT. Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro‐apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.