Cholesterol depletion by methyl-beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons

We recently demonstrated that although cholera toxin (CT) is found in detergent-insoluble domains/rafts at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism. Thus, cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) did not affect the rate of CT internalization from the plasma membrane, but did affect the rate of CT degradation, which occurs in lysosomes. In the current study, we analyze which step of CT intracellular transport is inhibited by MbetaCD. Whereas pre-incubation with MbetaCD completely blocked CT degradation, it had no effect on the degradation of wheat germ agglutinin (WGA) or bovine serum albumin (BSA), which are internalized by receptor-mediated and fluid phase endocytosis, respectively. Brefeldin A also completely blocked CT degradation but had no effect on WGA or BSA degradation. In contrast, MbetaCD did not affect CT degradation, or CT-mediated cAMP generation, when added to neurons after CT had been transported to the Golgi apparatus. We conclude that CT transport from endosomes to the Golgi apparatus is cholesterol-dependent, whereas CT transport from the Golgi apparatus to lysosomes is cholesterol-independent.     

Hyperthermia stimulates energy-proteasome-dependent protein degradation in cultured myotubes

Previous studies suggest that elevated temperature stimulates protein degradation in skeletal muscle, but the intracellular mechanisms are not fully understood. We tested the role of different proteolytic pathways in temperature-dependent degradation of long- and short-lived proteins in cultured L6 myotubes. When cells were cultured at different temperatures from 37 to 43 degrees C, the degradation of both classes of proteins increased, with a maximal effect noted at 41 degrees C. The effect of high temperature was more pronounced on long-lived than on short-lived protein degradation. By using blockers of individual proteolytic pathways, we found evidence that the increased degradation of both long-lived and short-lived proteins at high temperature was independent of lysosomal and calcium-mediated mechanisms but reflected energy-proteasome-dependent degradation. mRNA levels for enzymes and other components of different proteolytic pathways were not influenced by high temperature. The results suggest that hyperthermia stimulates the degradation of muscle proteins and that this effect of temperature is regulated by similar mechanisms for short- and long-lived proteins. Elevated temperature may contribute to the catabolic response in skeletal muscle typically seen in sepsis and severe infection.     

Helix pomatia agglutinin binds specifically to the Golgi apparatus in cultured human fibroblasts and reveals two Golgi apparatus-specific glycoproteins

Fluorochrome-coupled Helix pomatia agglutinin (HPA), but not other lectin-conjugates with the same nominal specificity, bound specifically to the Golgi apparatus in cultured human fibroblasts, revealing a cytoplasmic juxtanuclear reticular structure. Unlike other Golgi-binding lectins the HPA-conjugates did not bind to the cell surface membrane or pericellular matrix. Experiments with 35S-methionine-labeled cells showed that HPA recognized two glycoproteins of Mr 170,000 and 400,000 among the secreted products of fibroblasts and two major cellular glycoproteins of Mr 40,000 and Mr 180,000 in Triton X-100 extracts of the cells. The two cellular HPA-binding polypeptides were also found in cells depleted of secretory products and in cells pulse-labeled shortly with 35S-methionine and then chased with methionine containing medium up to 12 h. These findings suggest that the two cellular glycoproteins recognized by HPA are retained in the Golgi apparatus and are therefore not precursors of secretory proteins. The results suggest that there are two endogenous, Golgi apparatus-specific glycoproteins in cultured human fibroblasts with terminal non-reducing O-glycosidic N-acetyl galactosaminyl residues.     

Novel monoclonal antibody mAb G3A5 recognizes 138-kDa glycoprotein localized on the Golgi membrane.

Partially purified Golgi membranes of HeLa cells were used as antigen to produce a novel monoclonal antibody (mAb G3A5). The mAb G3A5 specifically labeled Golgi apparatus of human and monkey cultured cells as ascertained by indirect immunofluorescence but did not stain those of bovine or mouse cells. Treatment with nocodazole and brefeldin A (BFA) induced fragmentation and redistribution of the staining. Western immunoblot analysis showed that mAb G3A5 was directed against a single polypeptide with an apparent molecular mass of 138-kDa (p138 antigen). The p138 antigen is an integral membrane protein of the Golgi apparatus, as assessed by several assays: protease protection, salt wash and flotation in sucrose density gradient centrifugation. The p138 antigen was purified using immunoaffinity chromatography. The apparent molecular mass of the p138 antigen decreased by 2 to 4 kDa after treatment with the peptide: N-glycosidase F, while digestion with ENDO F or Neuraminidase did not have this effect. Thus, p138 antigen is a glycoprotein containing asparagine-linked carbohydrates.     

Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum

The tyrosine kinase Src is present on the Golgi membranes. Its role, however, in the overall function and organization of the Golgi apparatus is unclear. We have found that in a cell line called SYF, which lacks the three ubiquitous Src-like kinases (Src, Yes, and Fyn), the organization of the Golgi apparatus is perturbed. The Golgi apparatus is composed of collapsed stacks and bloated cisternae in these cells. Expression of an activated form of Src relocated the KDEL receptor (KDEL-R) from the Golgi apparatus to the endoplasmic reticulum. Other Golgi-specific marker proteins were not affected under these conditions. Because of the specific effect of Src on the location of KDEL-R, we tested whether protein transport between ER and the Golgi apparatus involves Src. Transport of Pseudomonas exotoxin, which is transported to the ER by binding to the KDEL-R is accelerated by inhibition or genetic ablation of Src. Protein transport from ER to the Golgi apparatus however, is unaffected by Src deletion or inhibition. We propose that Src has an appreciable role in the organization of the Golgi apparatus, which may be linked to its involvement in protein transport from the Golgi apparatus to the endoplasmic reticulum.     

The mitochondrial probe rhodamine 123 inhibits in isolated hepatocytes the degradation of short-lived proteins

The fluorescent dye rhodamine 123 (R123) decreases the intracellular ATP levels and also inhibits the degradation of short-lived proteins in isolated hepatocytes. This inhibition affects lysosomal and, to some extent, non-lysosomal mechanisms. The degradation of short-lived proteins decreases more when ATP levels are less than 40% of those in control cells, in contrast to the reported linear correlation between ATP levels and degradation of long-lived proteins. R123 provides a powerful probe for clarifying the proteolytic mechanisms involved in degradation of short-lived proteins and the ATP requirements in protein degradation. Indeed, as illustrated, the results suggest different mechanisms for the degradation of short- and long-lived proteins. Moreover, they provide a warning for the clinical use of this reagent.     

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.     

Oligomerization and intracellular protein transport: dimerization of intestinal dipeptidylpeptidase IV occurs in the Golgi apparatus.

It was postulated that newly synthesized membrane proteins need to be assembled into oligomers in the endoplasmic reticulum in order to be transported to the Golgi apparatus. By use of the differentiated human adenocarcinoma cell line Caco-2, the general validity of this proposal was studied for small intestinal brush border enzymes which are dimers in most mammalian species. Chemical cross-linking experiments and sucrose gradient rate-zonal centrifugation revealed that dipeptidylpeptidase IV is present as a dimer in the brush border membrane of Caco-2 cells whereas the disaccharidase sucrase-isomaltase appears to be a monomer. Dipeptidylpeptidase IV was found to dimerize immediately after complex glycosylation, an event associated with the Golgi apparatus. Dimerization of this enzyme was inhibited by CCCP but did not depend on complex glycosylation of N-linked carbohydrates as assessed by the use of the trimming inhibitor 1-deoxymannojirimycin. It is concluded that dimerization of dipeptidylpeptidase IV occurs in a late Golgi compartment and therefore cannot be a prerequisite for its export from the endoplasmic reticulum.     

Characterization of the inhibition of protein degradation by insulin in L6 cells

In muscle cells, protein degradation occurs by lysosomal and nonlysosomal mechanisms but the mechanism by which insulin inhibits protein degradation is not well understood. Using cultured L6 myotubes, the effect of insulin on muscle cell protein degradation was examined. Cells were labeled for 18 h with [3H]leucine or [3H]tyrosine and protein degradation measured by release of TCA-soluble radioactivity. Incubation with insulin for 0.5, 1, 2, or 3 h produced 0, 6, 12, and 13% inhibition, respectively, at 10(-7) M. If the cells were incubated for 3 h prior to the addition of insulin to remove short-lived proteins, the effect of insulin was enhanced, producing 26% inhibition. Very long-lived protein degradation (cells labeled for 48 h, chased for 24 h before the addition of insulin) was only inhibited 17% by insulin. This was due to serum starvation during the chase since the addition of serum to the chase medium produced a subsequent inhibition of 38% by insulin. Thus insulin had a greater effect on the degradation of longer-lived proteins. Use of inhibitors suggested that insulin requires internalization and degradation to produce inhibition of protein degradation and acts through both the proteasome and lysosomes. There appears to be no interaction with the calpains.     

The Golgi localization of Arabidopsis thaliana β1,2-xylosyltransferase in plant cells is dependent on its cytoplasmic and transmembrane sequences

To investigate the targeting of proteins to the plant Golgi we studied Arabidopsis thaliana 1,2-xylosyltransferase (XylT), a glycosyltransferase which is unique to plants and some invertebrates. Different deletion constructs of the putative cytoplasmic (C)-transmembrane (T)-stem (S) region of the enzyme were transiently expressed in the tobacco-related model plant species Nicotiana benthamiana. Subcellular localization of fusion proteins between CTS, CT, T, or C domains and the reporter molecule green fluorescent protein by fluorescence microcopy and density-gradient centrifugation revealed that the CT region alone is sufficient to sustain Golgi retention of XylT without the contribution of any luminal sequences. The finding of an incomplete retention by the T region alone suggests an important auxiliary role of the C domain in Golgi retention of the protein. However, the C segment did not confer any Golgi retention by itself, as the respective fusion protein was found exclusively in the cytoplasm. These results provide evidence that plant and mammalian cells rely on similar mechanisms to deliver glycosyltransferases to the Golgi apparatus.     

The degradation of endogenous and exogenous proteins in cultured smooth muscle cells

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards ¹²⁵I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [³H]phenylalanine, was reduced by only 10–17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (E_a = 18 kcal/mol) and short-lived proteins (E_a = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37°C in contrast to the breakdown of LDL which ceased below 20°C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.     

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors

This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles — transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles.     

Retrieval of HDEL proteins is required for growth of yeast cells

The ERD2 gene of Saccharomyces cerevisiae encodes the receptor which retrieves HDEL-containing containing ER proteins from the Golgi apparatus. Viable erd2 mutants have been isolated that show no obvious HDEL-dependent retention of the luminal ER protein BiP, suggesting that retrieval of HDEL proteins is not essential for growth. However, cells that lack Erd2p completely have a defective Golgi apparatus and cannot grow. This observation led to the suggestion that the receptor had a second function, possibly related to its ability to recycle from Golgi to ER. In this paper we investigate the requirements for Erd2p to support growth. We show that mutations that block its recycling also prevent growth. In addition, we show that all mutant receptors that can support growth have a residual ability to retrieve BiP, which is detectable when they are overexpressed. Mere recycling of an inactive form of the receptor, mediated by a cytoplasmic KKXX sequence, is not sufficient for growth. Furthermore, saturation of the receptor by expression of an HDEL-tagged version of pro-alpha factor inhibits growth, even of strains that do not show obvious BiP retention. We conclude that growth requires the HDEL-dependent retrieval of one or more proteins, and that these proteins can be recognized even under conditions where BiP is secreted. Genetic screens have failed to identify any one protein whose loss could account for the Erd2p requirement. Therefore, a growth may require the retention of multiple HDEL proteins in the ER, or alternatively the removal of such proteins from the Golgi apparatus.     

Studies of the sites of intracellular degradation of apolipoprotein B in Hep G2 cells.

We previously reported that treatment of Hep G2 cells with oleate significantly increased apolipoprotein B (apoB) secretion by reducing early intracellular degradation of nascent apoB. In the current study, inhibitors of secretory protein transport (brefeldin A and monensin), cell fractionation studies, and protease protection assays were utilized to determine the location of apoB degradation and to better define the mechanism whereby oleate treatment reduces nascent apoB intracellular degradation. When cells were treated with brefeldin A, which blocks endoplasmic reticulum (ER) to Golgi protein transport, apoB degradation continued in control cells, suggesting that apoB is degraded in the ER. When oleate-treated cells were blocked with brefeldin A, oleate failed to protect apoB from intracellular degradation. The effects of brefeldin A were not due to effects on lipid synthesis as brefeldin A did not inhibit the synthesis of triglyceride, phospholipid, free cholesterol, or cholesteryl ester in control cells and did not prevent the increases in triglyceride (14-fold) and phospholipid (1.4-fold) synthesis seen in oleate-treated cells. Simultaneous treatment of cells with brefeldin A and nocodazole, which inhibits retrograde transport of proteins from Golgi to ER, added to the evidence for the ER as the site of apoB degradation. This conclusion received further support from experiments in which cells were treated with monensin, a Na+ ionophore which halts protein secretion at the level of the trans-Golgi network. Early degradation of nascent apoB (between 10 and 20 min of chase) was observed in monensin-treated cells, but then cellular apoB degradation ceased and apoB was stable during the remaining chase period. More apoB accumulated in the Golgi of cells that had been treated with oleate and monensin. These results suggest that ER degradation occurs in monensin-treated cells, but then stops as apoB is transferred to the Golgi. The results obtained in whole cells were confirmed in studies using isolated ER and Golgi, which indicated that ER contains a proteolytic activity which degrades apoB, in vitro, whereas Golgi does not. ApoB degradation in isolated ER was not reduced by pretreatment with oleate. Finally, protease protection assays carried out with isolated microsomes indicated that a majority of the apoB in both control or oleate-treated HepG2 cells was located on the cytosolic side of the membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Degradation of short-lived proteins is decreased by centrifugation

We have examined the effects of enucleation and of inhibitors of mRNA synthesis (actinomycin D and cordycepin) on protein turnover of HeLa cells. Enucleation markedly inhibited the rate of protein degradation for short-lived proteins. However, cells centrifuged in the absence of cytochalasin B at the speed required to obtain cytoplasts showed protein degradation rates identical to those of cytoplasts, while inhibitors of mRNA synthesis did not affect the process. Although enucleation may affect degradation of specific proteins, these results suggest that centrifugation is largely responsible for the inhibition of protein degradation in cytoplasts.     

Phosphoinositide-regulated retrograde transport of ricin: crosstalk between hVps34 and sorting nexins

The plant toxin ricin is transported from the plasma membrane via early endosomes and the Golgi apparatus to the endoplasmic reticulum. From this compartment, it enters the cytosol and inhibits protein synthesis. Lipid phosphorylation is an important regulator of vesicular transport, and in the present study we have investigated the role of the phosphatidylinositol (PI) 3-kinase hVps34 in retrograde transport of ricin. Our data demonstrate that transport of ricin from endosomes to the Golgi apparatus in human embryonic kidney cells (HEK 293) is dependent on PI(3)P. By using PI 3-kinase inhibitors, by sequestering the hVps34 product PI(3)P and by expressing mutants of hVps34 or small interfering RNA targeted against its messenger RNA, we show that hVps34 and its product PI(3)P are involved in transport of ricin from endosome to Golgi apparatus. Furthermore, we identify two effector proteins in the hVps34-dependent pathway, namely sorting nexin (SNX) 2 and SNX4. Knockdown of SNX2 or SNX4 inhibits ricin transport to the Golgi apparatus to the same extent as when hVps34 is perturbed. Furthermore, inhibition or knockdown of hVps34 redistributes these proteins. Interestingly, knocking down both SNX2 and SNX4 results in a better inhibition than knocking down only one of them, suggesting that they may act on separate pathways.     

Dicumarol, an inhibitor of ADP-ribosylation of CtBP3/BARS, fragments golgi non-compact tubular zones and inhibits intra-golgi transport

Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex.     

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.     

Membrane protein transport between the endoplasmic reticulum and the Golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching

The mechanisms that control protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus are poorly characterized in plants. Here, we examine in tobacco leaves the structural relationship between Golgi and ER membranes using electron microscopy and demonstrate that Golgi membranes contain elements that are in close association and/or in direct contact with the ER. We further visualized protein trafficking between the ER and the Golgi using Golgi marker proteins tagged with green fluorescent protein. Using photobleaching techniques, we showed that Golgi membrane markers constitutively cycle to and from the Golgi in an energy-dependent and N-ethylmaleimide-sensitive manner. We found that membrane protein transport toward the Golgi occurs independently of the cytoskeleton and does not require the Golgi to be motile along the surface of the ER. Brefeldin A treatment blocked forward trafficking of Golgi proteins before their redistribution into the ER. Our results indicate that in plant cells, the Golgi apparatus is a dynamic membrane system whose components continuously traffic via membrane trafficking pathways regulated by brefeldin A- and N-ethylmaleimide-sensitive machinery.