Retention of membrane proteins by the endoplasmic reticulum

We have used a monoclonal antibody specific for a hydrocarbon-induced cytochrome P450 to localize, by electron microscopy, the epitope- specific cytochrome P450. The cytochrome was found in the rough and smooth endoplasmic reticulum (ER) and the nuclear envelope of hepatocytes. Significant quantities of cytochrome P450 were not found in Golgi stacks. We also could not find any evidence of Golgi- associated processing of the Asn-linked oligosaccharide chains of two well-characterized ER membrane glycoprotein enzymes (glucosidase II and hexose-6-phosphate dehydrogenase), or of the oligosaccharides attached to the bulk of the glycoproteins of the ER membrane. We conclude that these ER membrane proteins are efficiently retained during a process of highly selective export from this organelle.     

Topogenesis of membrane proteins at the endoplasmic reticulum

Most eukaryotic membrane proteins are cotranslationally integrated into the endoplasmic reticulum membrane by the Sec61 translocation complex. They are targeted to the translocon by hydrophobic signal sequences, which induce the translocation of either their N- or their C-terminal sequence. Signal sequence orientation is largely determined by charged residues flanking the apolar sequence (the positive-inside rule), folding properties of the N-terminal segment, and the hydrophobicity of the signal. Recent in vivo experiments suggest that N-terminal signals initially insert into the translocon head-on to yield a translocated N-terminus. Driven by a local electrical potential, the signal may invert its orientation and translocate the C-terminal sequence. Increased hydrophobicity slows down inversion by stabilizing the initial bound state. In vitro cross-linking studies indicate that signals rapidly contact lipids upon entering the translocon. Together with the recent crystal structure of the homologous SecYEbeta translocation complex of Methanococcus jannaschii, which did not reveal an obvious hydrophobic binding site for signals within the pore, a model emerges in which the translocon allows the lateral partitioning of hydrophobic segments between the aqueous pore and the lipid membrane. Signals may return into the pore for reorientation until translation is terminated. Subsequent transmembrane segments in multispanning proteins behave similarly and contribute to the overall topology of the protein.     

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.     

Lipid metabolism and structure-activity features of the endoplasmic reticulum membrane in regenerating rat liver]

The relationship between the neutral lipid and phospholipid metabolism and some structure-function peculiarities of regenerating rat liver endoplasmic reticulum membranes (13 hours after surgery, i.e., corresponding to the G1-period of the cell cycle) was studied. There was an increase in the degree of the endoplasmic reticulum membrane development and the nonesterified fatty acid (NFA) and triglyceride (TG) content in regenerating rat liver microsomes. The relative specific radioactivity of neutral lipid and phospholipid fractions in regenerating rat liver microsomes was lower than in control animals, presumably due to the high rate of the microsomal lipid exchange in the regenerating liver with other cell organelles. The changes in the lipid content and rate of their metabolism in the regenerating rat liver were associated with the increase in the membrane microviscosity and the decrease in the activity of the membrane-bound enzyme (glucose-6-phosphatase). The differences in the time-dependent changes in the synthesis and metabolism of lipids in the NFA and TG fractions may be regarded as an endogenous factor determining the structure-function peculiarities of endoplasmic reticulum membranes.     

Cryoresistance of rat liver endoplasmic reticulum membranes]

The effects of freezing of microsomes in liquid nitrogen and those of storage of microsomal suspensions at 2-4 degrees C and -3 - -5 degrees C for 24 hrs, on the enzymatic activities and hydrophobicity of membranes were studied. The hydrophobicity was determined by fluorescence of bound 1,8-anilino-naphthalene sulfonate. Rapid freezing of the microsomal suspension in liquid nitrogen followed by rapid warming did not change the hydrophobicity of the membranes, the rate of enzymatic lipid peroxidation, the level of cytochrome P-450 and the activity of NADH- and NADPH-cytochrome c reductase. A considerable decrease in the rate of enzymatic lipid peroxidation and membrane hydrophobicity was observed in the microsomes stored for 24 hrs at 2-4 degrees C. The 24-hr storage at -3 - -5 degrees C with subsequent thawing resulted in a rapid aggregation of the microsomes.     

The separation of rat liver endoplasmic reticulum membrane proteins by two dimensional polyacrylamide gel electrophoresis

The separation of rat liver endoplasmic reticulum membrane proteins by two dimensional polyacrylamide gel electrophoresis is described. By this method, the proteins of the rough membrane ribosomes could be separated from the other rough membrane proteins. Both rough and smooth membrane fractions contain at least 30 defined membranal proteins. The electrophoretic patterns of rough and smooth membrane proteins are clearly different.     

A class of membrane proteins shaping the tubular endoplasmic reticulum

How is the characteristic shape of a membrane bound organelle achieved? We have used an in vitro system to address the mechanism by which the tubular network of the endoplasmic reticulum (ER) is generated and maintained. Based on the inhibitory effect of sulfhydryl reagents and antibodies, network formation in vitro requires the integral membrane protein Rtn4a/NogoA, a member of the ubiquitous reticulon family. Both in yeast and mammalian cells, the reticulons are largely restricted to the tubular ER and are excluded from the continuous sheets of the nuclear envelope and peripheral ER. Upon overexpression, the reticulons form tubular membrane structures. The reticulons interact with DP1/Yop1p, a conserved integral membrane protein that also localizes to the tubular ER. These proteins share an unusual hairpin topology in the membrane. The simultaneous absence of the reticulons and Yop1p in S. cerevisiae results in disrupted tubular ER. We propose that these "morphogenic" proteins partition into and stabilize highly curved ER membrane tubules.     

Integration of membrane proteins into the endoplasmic reticulum requires GTP

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.     

Transmembrane domain-dependent partitioning of membrane proteins within the endoplasmic reticulum

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein-protein and protein-lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail-anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER-Golgi boundary by simple receptor-independent mechanisms based on partitioning.     

Identification of the endoplasmic reticulum targeting signal in vesicle-associated membrane proteins

The vesicle-associated membrane proteins (Vamp(s)) function as soluble N-ethylmaleimide-sensitive factor attachment receptor proteins in the intracellular trafficking of vesicles. The membrane attachment of Vamps requires a carboxyl-terminal hydrophobic sequence termed an insertion sequence. Unlike other insertion sequence-containing proteins, targeting of the highly homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP and a membrane-bound receptor. To determine if this mechanism of targeting to the endoplasmic reticulum extends to other Vamps, we compared the membrane binding of Vamp1 and Vamp2 with the distantly related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and a membrane component to target to the endoplasmic reticulum. Furthermore, binding curves for the three Vamps overlap, suggesting a common receptor-mediated process. We identified a minimal endoplasmic reticulum targeting domain that is both necessary and sufficient to confer receptor-mediated, ATP-dependent, binding of a heterologous protein to microsomes. Surprisingly, this conserved sequence includes four positively charged amino acids spaced along an amphipathic sequence, which unlike the carboxyl-terminal targeting sequence in mitochondrial Vamp isoforms, is amino-terminal to the insertion sequence. Because Vamps do not bind to phospholipid vesicles, it is likely that these residues mediate an interaction with a protein, rather than bind to acidic phospholipids. Therefore, we suggest that a bipartite motif is required for the specific targeting and integration of Vamps into the endoplasmic reticulum with receptor-mediated recognition of specifically configured positive residues leading to the insertion of the hydrophobic tail into the membrane.     

Targeting of rough endoplasmic reticulum membrane proteins and ribosomes in invertebrate neurons

The endoplasmic reticulum (ER) is divided into rough and smooth domains (RER and SER). The two domains share most proteins, but RER is enriched in some membrane proteins by an unknown mechanism. We studied RER protein targeting by expressing fluorescent protein fusions to ER membrane proteins in Caenorhabditis elegans. In several cell types RER and general ER proteins colocalized, but in neurons RER proteins were concentrated in the cell body, whereas general ER proteins were also found in neurites. Surprisingly RER membrane proteins diffused rapidly within the cell body, indicating they are not localized by immobilization. Ribosomes were also concentrated in the cell body, suggesting they may be in part responsible for targeting RER membrane proteins.     

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

Background  

Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms.     

Reconstitution of liver endoplasmic reticulum membranes from solubilized proteins and lipids by removal of detergent]

A method for membrane reconstitution from cholate-solubilized microsomal proteins and lipids by a removal of the detergent on a column with charcoal has been developed. A comparative study showed that the membranes reconstituted by a dialysis or absorption do not differ from each other in terms of membrane proteins incorporation into lipid vesicles and cytochrome P-450 reconversion into cytochrome P-450. A possibility of biomembrane reconstitution from membrane proteins and lipids solubilized by a non-ionic detergent Triton X-100 was shown. A removal of the detergent results in a formation of membranes, which are chemically close to the original ones but ultrastructurally very different from the latter. On the other hand, absorption or dialysis of cholate-solubilized proteins and lipids results in reconstituted membranes with asymmetrically arranged intramembrane particles located on the hydrophobic surfaces of the membrane halves. The number and size of these particles are similar to those of the original microsomal membranes.     

Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress and cell death in liver cells

Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein in response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin-sensitive calcium stores and increased biochemical markers of ER stress over similar time courses (6 h). These changes preceded cell death, which was only observed at later time points (16 h). Co-incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death observed in response to palmitate. Inclusion of calcium chelators, BAPTA-AM or EGTA, reduced palmitate- and stearate-mediated enrichment of cytochrome c in post-mitochondrial supernatant fractions and cell death. These data suggest that redistribution of ER luminal calcium contributes to long chain saturated fatty acid-mediated ER stress and cell death.     

Aluminum alters calcium transport in plasma membrane and endoplasmic reticulum from rat brain

Calcium is actively transported into intracellular organelles and out of the cytoplasm by Ca²⁺/Mg²⁺-ATPases located in the endoplasmic reticulum and plasma membranes. We studied the effects of aluminum on calcium transport in the adult rat brain. We examined ⁴⁵Ca-uptake in microsomes and Ca²⁺-ATPase activity in microsomes and synaptosomes isolated from the frontal cortex and cerebellum of adult male Long-Evans rats. ATP-dependent⁴⁵Ca-uptake was similar in microsomes from both brain regions. The addition of 50-800 μM AICI₃ resulted in a concentration-dependent inhibition of ⁴⁵Ca-uptake. Mg²⁺-dependent Ca²⁺-ATPase activity was significantly lower in synaptosomes compared to microsomes in both frontal cortex and cerebellum. In contrast to the uptake studies, AICI³ stimulated Mg²⁺-dependent Ca²⁺-ATPase activity in both microsomes and synaptosomes from both brain regions. To determine the relationship between aluminum and Mg²⁺, we measured ATPase activity in the presence of increasing concentrations of Mg²⁺ or AICI³. Maximal ATPase activity was obtained between 3 and 6 mM Mg²⁺. When we substituted AICI³ for Mg²⁺, ATPase activity was also stimulated in a concentration-dependent manner, but to a greater extent than with Mg²⁺. One interpretation of these data is that aluminum acts at multiple sites to displace both Mg²⁺ and Ca²⁺, increasing the activity of the Ca²⁺-ATPase, but disrupting transport of calcium.     

Contacts of the endoplasmic reticulum membrane with plasmalemma in plant cells

It is shown by electron microscopy that, in maize root cells, there is close contact between the membrane of the endoplasmic reticulum and the plasmalemma. A qualitative preliminary comparison is conducted between these contacts and high-permeability intercellular contacts in animals.