Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.     

Inheritance of the endoplasmic reticulum and Golgi apparatus

Yeast and mammalian cells use a variety of different mechanisms to ensure that the endoplasmic reticulum and Golgi apparatus are inherited by both daughter cells on cell division. In yeast, endoplasmic reticulum inheritance involves both active microtubule and passive actin-based mechanisms, while the Golgi is transported into the forming daughter cell by an active actin-based mechanism. Animal cells actively partition the endoplasmic reticulum and Golgi apparatus, but association with the mitotic spindle-rather than the actin cytoskeleton-appears to be the mechanism     

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.     

Bidirectional membrane traffic between the endoplasmic reticulum and Golgi apparatus

Membrane traffic between the endoplasmic reticulum and Golgi apparatus is a highly regulated process that uses distinct anterograde and retrograde pathways. These pathways link two organelles that together function as a dynamic membrane system specialized for the biosynthesis and sorting of membrane to be used throughout the cell. The nature and underlying biochemical control of membrane transport along these pathways is thought to be tied to a common regulatory system involving assembly and disassembly of cytosolic proteins on membranes.     

Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

Trafficking and sorting of lipids during transport from the endoplasmic reticulum to the Golgi apparatus was studied using a cell-free system from rat liver. Transitional elements of the endoplasmic reticulum were prepared from liver slices prelabeled with [14C]- or [3H]acetate as the donor fraction. Non-radioactive Golgi apparatus were immobilized on nitrocellulose as the acceptor. When reconstituted, the radiolabeled donor retained a capacity to transfer labeled lipids to the non-radioactive Golgi apparatus acceptor. Transfer exhibited two kinetically different components. One was stimulated by ATP, facilitated by cytosol and inhibited by guanosine 5'-O-(thiotriphosphate) and N-ethylmaleimide. In parallel with protein transport, the ATP-dependent lipid transfer occurred with a temperature transition at about 20 degrees C. The other was not stimulated by ATP, did not require cytosol, was acceptor unspecific, was unaffected by inhibitors and, while temperature dependent, did not exhibit a sharp temperature transition. The ATP-independent transfer was non-vesicular. In contrast, the ATP-dependent transfer was vesicular. Transition vesicles isolated by preparative free-flow electrophoresis, when used as the donor fraction, transferred lipids to Golgi apparatus acceptor with a 5-6-fold greater efficiency than that exhibited by the unfractionated transitional endoplasmic reticulum. Formation of transition vesicles was ATP-dependent. Transferred lipids were chiefly phosphatidylcholine and cholesterol. Membrane triglycerides, major constituents of the transitional endoplasmic reticulum membranes, were both depleted in the transition vesicle-enriched fractions and not transferred to Golgi apparatus suggestive of lipid sorting prior to or during transition vesicle formation. The characteristics of the ATP plus cytosol-dependent transfer were similar to those for protein transfer mediated by transition vesicles. Thus, the 50-70-nm vesicles derived from transitional endoplasmic reticulum appear to function in the trafficking of both newly synthesized proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.     

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.     

Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.     

Flow kinetics of a nucleoside phosphatase common to endoplasmic reticulum, Golgi apparatus, and plasma membrane of rat liver

Nucleoside mono-, di- and triphosphatase activities of highly purified endoplasmic reticulum (ER), Golgi apparatus, and plasma membrane fractions of rat liver were compared. The highest rates of hydrolysis were always in ER or plasma membrane. Golgi apparatus activity was intermediate between those of ER and plasma membrane. This relationship was true for both freshly isolated fractions and salt-extracted membranes. Detergent solubilization of the membranes, polyacrylamide gel electrophoresis of the solubilized proteins, and localization of the enzyme activities on the gel revealed bands of enzyme activity which had identical mobilities in all three membrane fractions as well as other bands of activity that occurred only in ER and to a lesser degree in the Golgi apparatus. Antibodies raised against one of the phosphatase bands of plasma membrane which was common to all three membrane fractions cross-reacted with the corresponding phosphatase band in ER and Golgi apparatus. The anti-nucleoside phosphatase was utilized in combination with pulse-chase techniques to investigate the flow kinetics of transfer of newly synthesized enzyme among different cell compartments. Label first appeared in nucleoside phosphatase within the ER. Maximum specific activity was observed at about 5 min after injection of label and was followed by rapid loss of label. This was followed by appearance of label in Golgi apparatus 15 to 25 min after injection of label and by subsequent rapid loss of label. Plasma membranes were labeled last with no evidence of either rapid accumulation of label or of rapid turnover. Flow of nucleoside phosphatase from its site of synthesis and insertion into the membrane at the endoplasmic reticulum to the plasma membrane via the Golgi apparatus is indicated but in a manner whereby a significant fraction of the protein may be processed (removed?) from the membrane concomitant with the flow process.     

Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond

Significant advances have been made in recent years that have increased our understanding of the trafficking to and from membranes that are functionally linked to the Golgi apparatus in plants. New routes from the Golgi to organelles outside the secretory pathway are now being identified, revealing the importance of the Golgi apparatus as a major sorting station in the plant cell. This review discusses our current perception of Golgi structure and organization as well as the molecular mechanisms that direct traffic in and out of the Golgi.     

The Golgi apparatus maintains its organization independent of the endoplasmic reticulum

Under artificial conditions Golgi enzymes have the capacity to rapidly accumulate in the endoplasmic reticulum (ER). These observations prompted the idea that Golgi enzymes constitutively recycle through the ER. We have tested this hypothesis under physiological conditions through use of a procedure that captures Golgi enzymes in the ER. In the presence of rapamycin, which induces a tight association between FKBP (FK506-binding protein) and FRAP (FKBP-rapamycin-associated protein), an FKBP-tagged Golgi enzyme can be trapped when it visits the ER by an ER-retained protein fused to FRAP. We find that although FKBP-ERGIC-53 of the ER-Golgi intermediate compartment (ERGIC) rapidly cycles through the ER (30 min), FKBP-Golgi enzyme chimeras remain stably associated with Golgi membranes. We also demonstrate that Golgi dispersion upon nocodazole treatment mainly occurs through a mechanism that does not involve the recycling of Golgi membranes through the ER. Our findings suggest that the Golgi apparatus, as defined by its collection of resident enzymes, exists independent of the ER.     

Retinoid modulation of cell-free membrane transfer between endoplasmic reticulum and Golgi apparatus

Cell-free transfer of radiolabeled membrane proteins from part-rough, part-smooth transitional elements of the endoplasmic reticulum to Golgi apparatus immobilized to nitrocellulose in the presence of nucleoside triphosphate, an ATP-regenerating system and a cytosol fraction was promoted by retinol. At an optimum concentration of 1 microgram/ml, the rate and amount of transfer was approximately doubled over 1 to 2 h of incubation in the cell-free system. The transition vesicles induced to form in the cell-free system were concentrated by preparative free-flow electrophoresis in order to study separately the steps of vesicle formation from transitional endoplasmic reticulum and the steps of vesicle fusion with Golgi apparatus. The retinol effect was on vesicle formation as evidenced by an approx. 2-fold increase in transition vesicle numbers, as determined by electron microscope morphometry, and amount from protein determinations on the isolated fractions enriched in transition vesicles. The retinol response in the complete transfer could be eliminated by addition of concentrated cytosol, including cytosol depleted of retinol. An interaction of retinol with some component of the vesicle formation process, possibly involving guanine nucleotides, is indicated.     

Acyl transfer reactions associated with cis Golgi apparatus of rat liver

Isolated Golgi apparatus, highly purified from rat liver, were found to contain an acyl transfer activity capable of restoring the acyl chains of the lysophospholipid products of the action of phospholipase A₂ on phosphatidylcholine. The activity was located primarily in cis and medial Golgi apparatus fractions, had a pH optimum of 6.0 to 7.5 and was stimulated by various acyl-CoA derivatives but not by fatty acids plus ATP. The activity, determined from the conversion of [¹⁴C]lysophosphatidylcholine to [¹⁴C]phosphatidylcholine, was unaffected by EGTA, inhibited by manoalide at high concentrations (0.2 mM), and temperature-dependent. Temperature dependency, however, showed no definite transition temperature over the range 15 to 37°C. The results demonstrated that cis Golgi apparatus membranes have the enzymatic capacity to restore fatty acids lost from phospholipids through the action of phospholipase A. The latter has been previously suggested to occur at the cis Golgi apparatus membranes based on analyses of cell-free transfer of radiolabeled phosphatidylcholine.     

Isolation of plasma membrane,Golgi apparatus,and endoplasmic reticulum fractions from single homogenates of mouse liver

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K⁺-stimulated p-nitrophenyl phosphatase and (Na⁺ K⁺) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.     

Impact on the endoplasmic reticulum and Golgi apparatus of turnip mosaic virus infection

The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.     

Dynamic nucleation of Golgi apparatus assembly from the endoplasmic reticulum in interphase hela cells

Models of Golgi apparatus biogenesis and maintenance are focused on two possibilities: one is self-assembly from the endoplasmic reticulum, and the other is nucleation by a stable template. Here, we asked in three different experimental situations whether assembly of the Golgi apparatus might be dynamically nucleated. During microtubule depolymerization, the integral membrane protein p27 and the peripheral Golgi protein GM130, appeared in newly formed, scattered Golgi elements before three different Golgi apparatus cisternal enzymes, whereas GRASP55, a medial peripheral Golgi protein, showed, if anything, a tendency to accumulate in scattered Golgi elements later than a cisternal enzyme. During Golgi formation after brefeldin A washout, endoplasmic reticulum exit of Golgi resident enzymes could be completely separated from that of p27 and GM130. p27 and GM130 accumulation was onto newly organized perinuclear structures, not brefeldin A remnants, and preceded that of a cisternal enzyme. Reassembly was completely sensitive to guanosine 5'-diphosphate-restricted Sar1p. When cells were microinjected with Sar1pWT DNA to reverse a guanosine 5'-diphosphate-restricted Sar1p endoplasmic reticulum-exit block phenotype, GM130 and p27 collected perinuclearly with little to no exit of a cisternal enzyme from the endoplasmic reticulum. The overall data strongly indicate that the assembly of the Golgi apparatus can be nucleated dynamically by GM130/p27 associated structures. We define dynamic nucleation as the first step in a staged organelle assembly process in which new component association forms a microscopically visible structure onto which other components add later, e.g. Golgi cisternae.