Cell-free fusion of endocytic vesicles is regulated by phosphorylation

Okadaic acid and microcystin-LR, both potent inhibitors of protein phosphatases (PP), blocked vesicle fusion in a cell-free system. The effect of okadaic acid was reversed by the purified catalytic subunit of PP2A, but not PP1. Inhibition was gradual, required Mg-ATP, and was reduced by protein kinase inhibitors, indicating that it was mediated via protein phosphorylation. A candidate protein kinase would be cdc2 kinase, which normally is active in mitotic extracts and has been shown to inhibit endocytic vesicle fusion (Tuomikoski, T., M.-A. Felix, M. Dorée, and J. Gruenberg. 1989. Nature (Lond.). 342:942-945). However, it would appear that cdc2 kinase is not responsible for inhibition by okadaic acid. When compared to cytosol prepared from mitotic cells, okadaic acid did not increase cdc2 kinase activity sufficiently to account for the inhibition. In addition, inhibition was maintained when cdc2 protein was depleted from cytosol.     

Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle.

Phosphatase inhibitors microcystin-LR, tautomycin, and okadaic acid caused contraction and increased 20-kDa myosin light chain (MLC20) phosphorylation in Ca(2+)-free solutions in both phasic and tonic smooth muscle permeabilized with beta-escin, and inhibited the heavy meromyosin (HMM) phosphatase activity of smooth muscle homogenates with the same potency sequence: microcystin-LR greater than tautomycin greater than okadaic acid. The sensitivity to all three inhibitors was significantly higher, the half-times of relaxation and dephosphorylation were 4-6 times longer, and the HMM phosphatase and MLC20 kinase activity/smooth muscle cell wet weight was 2.0- and 1.9-fold lower in the tonic, femoral artery, than in the phasic, ileum or portal vein, smooth muscle. Preincubation with 0.2 microM inhibitor-2 decreased the HMM phosphatase activity by 35% in the ileum and by 60% in the femoral artery. The results suggest that the HMM phosphatases of smooth muscle have properties common to type 1 protein phosphatases, but are inhibited only partially by high concentrations of inhibitor-2, and that the lower HMM phosphatase activity of tonic smooth muscle may contribute to its greater sensitivity to phosphatase inhibitors and its slower rate of relaxation.     

Microcystin-LR and okadaic acid-induced cellular effects: a dualistic response

Microcystins, potent heptapeptide hepatotoxins produced by certain bloom-forming cyanobacteria, are strong protein phosphatase inhibitors. They covalently bind the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A), thereby influencing regulation of cellular protein phosphorylation. The paralytic shellfish poison, okadaic acid, is also a potent inhibitor of these PPs. Inhibition of PP1 and PP2A has a dualistic effect on cells exposed to okadaic acid or microcystin-LR, with both apoptosis and increased cellular proliferation being reported. This review summarises the existing data on the molecular effects of microcystin-LR inhibition of PP1 and PP2A both in vivo and in vitro, and where possible, compares this to the action of okadaic acid.     

Constitutive secretion of serum albumin requires reversible protein tyrosine phosphorylation events in trans-Golgi

Serum albumin secretion from rat hepatocytes proceeds via the constitutive pathway. Although much is known about the role of protein tyrosine phosphorylation in regulated secretion, nothing is known about its function in the constitutive process. Here we show that albumin secretion is inhibited by the tyrosine kinase inhibitor genistein but relatively insensitive to subtype-selective inhibitors or treatments. Secretion is also blocked in a physiologically identical manner by the tyrosine phosphatase inhibitors pervanadate and bisperoxo(1,10-phenanthroline)-oxovanadate. Inhibition of either the kinase(s) or phosphatase(s) leads to the accumulation of albumin between the trans-Golgi and the plasma membrane, whereas the immediate precursor proalbumin builds up in a proximal compartment. The trans-Golgi marker TGN38 is rapidly dispersed under conditions that inhibit tyrosine phosphatase action, whereas the distribution of the cis-Golgi marker GM130 is insensitive to genistein or pervanadate. By using a specifically reactive biotinylation probe, we detected protein tyrosine phosphatases in highly purified rat liver Golgi membranes. These membranes also contain both endogenous tyrosine kinases and their substrates, indicating that enzymes and substrates for reversible tyrosine phosphorylation are normal membrane-resident components of this trafficking compartment. In the absence of perturbation of actin filaments and microtubules, we conclude that reversible protein tyrosine phosphorylation in the trans-Golgi network is essential for albumin secretion and propose that the constitutive secretion of albumin is in fact a regulated process. vesicular trafficking; liver; genistein; pervanadate     

Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts.

Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.     

H-Ras does not need COP I- or COP II-dependent vesicular transport to reach the plasma membrane

Although vesicular transport of the H-Ras protein from the Golgi to the plasma membrane is well known, additional trafficking steps, both to and from the plasma membrane, have also been described. Notably, both vesicular and nonvesicular transport mechanisms have been proposed. The initial trafficking of H-Ras to the plasma membrane was therefore examined in more detail. In untreated cells, H-Ras appeared at the plasma membrane more rapidly than a protein carried by the conventional exocytic pathway, and no H-Ras was visible on Golgi membranes in >80% of the cells. H-Ras was still able to reach the plasma membrane when COP II-directed transport was disrupted by two different mutant forms of Sar1, when COP I-mediated vesicular traffic from the endoplasmic reticulum to the Golgi was inhibited with brefeldin A, or when microtubules were disrupted by nocodazole. Although some H-Ras was present in the secretory pathway, protein that reached the membranes of the endoplasmic reticulum-Golgi intermediate compartment was unable to move further in the presence of nocodozale. These results identify an alternative mechanism for H-Ras trafficking that circumvents conventional COPI-, COPII-, and microtubule-dependent vesicular transport. Thus, H-Ras has two simultaneous but distinct means of transport and need not depend on vesicular trafficking for its delivery to the plasma membrane.     

Hepatocyte DNA Replication Is Abolished by Inhibitors Selecting Protein Phosphatase 2A Rather Than Phosphatase 1

Primary rat hepatocytes exposed to the phosphoprotein phosphatase (PP) inhibitors microcystin-LR and okadaic acid showed extensive surface protrusions and release of cell fragments, like cells in apoptosis. Microinjected microcystin fully reproduced these effects; the calculated intracellular concentration required for 50% effect being about 1 μM. The effects were counteracted by antagonists of calmodulin or of the multifunctional calmodulin-activated protein kinase II. The DNA replication of the epidermal growth factor-stimulated hepatocytes was nearly completely inhibited by okadaic acid at concentrations below those giving overt morphological effects. However, microcystin did not inhibit the DNA replication. Calmodulin antagonists counteracted the effect of okadaic acid on DNA replication. Microinjection of inhibitor-1 and inhibitor-2 (both directed against PP1) had no effect on DNA replication. Based on the known selectivity of okadaic acid for PP type 2A versus that of type 1, and the lack of such selectivity for microcystin, it is concluded that DNA replication is abolished by moderate inhibition of PP2A. Inhibition of PP1 did not impede DNA replication, suggesting that the two major liver phosphatases may have opposite roles in the regulation of hepatocyte DNA replication.     

Role of protein phosphorylation in TNF-induced apoptosis: Phosphatase inhibitors synergize with TNF to activate DNA fragmentation in normal as well as TNF-resistant U937 variants

This study examined the role of protein phosphorylation in TNF induction of apoptosis in several tumor cell lines by testing the effects of agents that either stimulate or inhibit protein phosphorylation. The serine-threonine phosphatase inhibitors, okadaic acid (OKA) and calyculin A (CLA), synergistically augmented TNF-induced apoptosis in several TNF-sensitive tumor cell lines including the U937 histiocytic lymphoma, the BT-20 mammary carcinoma, and the LNCap prostatic tumor cell line. Furthermore, the phosphatase inhibitors completely reversed the TNF resistance of a variant (U9-TR) derived from U937. CLA also inhibited phosphatase activity in cell-free extracts from both U937 and U9-TR at the same concentrations (0.4–2.0 nM) that it synergized with TNF. In contrast, TNF treatment of U937 cells did not result in inhibition of phosphatase activity mediated by protein phosphatase 1 (PP1) and PP2A in cell extracts. Since the phosphatase inhibitors are known to increase the overall levels of protein phosphorylation in cells, this suggested that TNF may act by stimulating protein kinase (PK) activity. This hypothesis was supported by the results of testing a panel of relatively specific protein kinase inhibitors. TNF activation of DNA fragmentation was blocked by a potent inhibitor of myosin light chain kinase (MLCK) but was unaffected by inhibitors of cAMP or cGMP-dependent PKs. We postulate that a defect in the activation of MLCK or possibly some other as yet unknown PK may be responsible for the TNF resistance of U9-TR. Furthermore, this resistance may be circumvented by promoting protein phosphorylation with the serine-threonine-dependent phosphatase inhibitors.     

Sucrose-phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves: Evidence from the effects of okadaic acid and microcystin

Sucrose-phosphate synthase (SPS) purified from spinach leaves harvested in the dark, was activated by mammalian protein phosphatase 2A (PP2A). Activation of SPS in a fraction from darkened spinach leaves was largely prevented by either okadaic acid or microcystin-LR (specific inhibitors of PP1 and PP2A), while inhibitor-2 (a PP1 inhibitor) or Mg²⁺ (essential for PP2C) were ineffective. In vivo, okadaic add and microcystin-LR prevented the light-induced activation of SPS and decreased sucrose biosynthesis and CO₂ fixation. It is concluded that PP2A is the major SPS phosphatase in spinach. This study is the first to employ microcystin-LR for modulating protein phosphorylation in vivo.     

Naringin-sensitive phosphorylation of plectin,a cytoskeletal cross-linking protein,in isolated rat hepatocytes

To identify phosphoproteins that might play a role in naringin-sensitive hepatocellular cytoskeletal disruption and apoptosis induced by algal toxins, hepatocyte extracts were separated by gel electrophoresis and immunostained with a phosphothreonine-directed antibody. Use of dilute (5%) polyacrylamide gels containing 6 m urea allowed the resolution of one very large (approximately 500-kDa) okadaic acid- and naringin-sensitive phosphoprotein, identified by tryptic fingerprinting, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and immunostaining as the cytolinker protein, plectin. The naringin-sensitive phosphorylation induced by okadaic acid and microcystin-LR probably reflected inhibition of a type 2A protein phosphatase, whereas the naringin-resistant phosphorylation induced by calyculin A, tautomycin, and cantharidin probably involved a type 1 phosphatase. Okadaic acid caused a collapse of the plectin-immunostaining bile canalicular sheaths and the general cytoskeletal plectin network into numerous medium-sized plectin aggregates. Inhibitors of protein kinase C, cAMP-dependent protein kinase, or Ca(2+)/calmodulin-dependent kinase II had moderate or no protective effects on plectin network disruption, whereas naringin offered 86% protection. Okadaic acid induced a naringin-sensitive phosphorylation of AMP-activated protein kinase (AMPK), the stress-activated protein kinases SEK1 and JNK, and S6 kinase. The AMPK-activating kinase (AMPKK) is likely to be the target of inhibition by naringin, the other kinases serving as downstream components of an AMPKK-initiated signaling pathway.     

Inhibitors of protein phosphatases 1 and 2A differentially prevent intrinsic and extrinsic apoptosis pathways

Inhibitors of serine/threonine protein phosphatases can inhibit apoptosis. We investigated which protein phosphatases are critical for this protection using calyculin A, okadaic acid, and tautomycin. All three phosphatase inhibitors prevented anisomycin-induced apoptosis in leukemia cell models. In vitro, calyculin A does not discriminate between PP1 and PP2A, while okadaic acid and tautomycin are more selective for PP2A and PP1, respectively. Increased phosphorylation of endogenous marker proteins was used to define concentrations that inhibited each phosphatase in cells. Concentrations of each inhibitor that prevented anisomycin-induced apoptosis correlated with inhibition of PP2A. The inhibitors prevented Bax translocation to mitochondria, indicating inhibition upstream of mitochondria. Tautomycin and calyculin A, but not okadaic acid, also prevented apoptosis induced through the CD95/Fas death receptor, and this protection correlated with inhibition of PP1. The inhibitors prevented Fas receptor oligomerization, FADD recruitment, and caspase 8 activation. The differential effects of PP1 and PP2A in protection from death receptor and mitochondrial-mediated pathways of death, respectively, may help one to define critical steps in each pathway, and regulatory roles for serine/threonine phosphatases in apoptosis.     

Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants

The cyclic heptapeptide, microcystin-LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with Ki values below 0.1 nM. Protein phosphatase 2B is inhibited 1000-fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin-LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin-LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin-LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.     

Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro

We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using digitonin-permeabilized cell assays to measure nuclear import and export, we found that the phosphatase inhibitors okadaic acid and microcystin inhibit transport mediated by the import receptors importin beta and transportin, but not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad specificity protein kinase inhibitor staurosporine, indicate that transport inhibition is due to elevated phosphorylation of a component of the nuclear transport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive component of the nuclear transport machinery also is present in permeabilized cells and is most likely a component of the nuclear pore complex. Substrate binding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphatase inhibitors, suggesting that transport inhibition by protein phosphorylation does not involve these steps. These results suggest that cells have mechanisms to negatively regulate entire nuclear transport pathways, thus providing a means to globally control cellular activity through effects on nucleocytoplasmic trafficking.     

Identification of a 42 kDa protein as a substrate of protein phosphatase 1 in cilia from Paramecium.

Okadaic acid, a specific inhibitor of protein phosphatase 1 in Paramecium causes sustained backward swimming in response to depolarising stimuli (S. Klumpp et al. (1990) EMBO J. 9, 685). Here, we employ okadaic acid, tautomycin, microcystin LR and inhibitor 1 as phosphatase inhibitors to identify a 42 kDa protein in the excitable ciliary membrane that is dephosphorylated by protein phosphatase 1. Identification of the 42 kDa protein was facilitated by the finding that the protein kinase responsible for its phosphorylation uses Ca-ATP as a substrate just as effectively as Mg-ATP. Notably, dephosphorylation of the 42 kDa protein is specifically inhibited by cyclic AMP; cyclic GMP has no effect.     

Differing effects of the protein phosphatase inhibitors okadaic acid and microcystin on translation in reticulocyte lysates.

The effects of the cyanobacterial toxin and protein phosphatase inhibitor, microcystin, on translation in rabbit reticulocyte lysates have been studied. Microcystin inhibited translation with similar potency to the protein phosphatase inhibitor okadaic acid. Unlike low concentrations of okadaic acid, however, it inhibited both the initiation and elongation stages. This was demonstrated using EGTA to inhibit the phosphorylation and inactivation of elongation factor eEF-2. A method for detecting changes in eEF-2 phosphorylation was developed. eEF-2 was found to exist as three different species: eEF-2 was largely monophosphorylated in reticulocyte lysates under control conditions, the remainder being unphosphorylated. Okadaic acid and microcystin increased the level of the bisphosphorylated species. The implications of multiple phosphorylation of eEF-2 for the control of translation is discussed. Microcystin was also found to increase the phosphorylation of eIF-2 alpha (and therefore to inhibit initiation) at lower concentrations than okadaic acid, suggesting that the major eIF-2 alpha phosphatase in the reticulocyte lysate is phosphatase-1.     

Mechanism of inhibition of insulin-stimulated glucose transport by 4-bromocrotonic acid in 3T3-L1 adipocytes

We have demonstrated previously that 4-bromocrotonic acid (Br-C4) inhibited insulin-stimulated glucose transport by interfering with GLUT4 translocation. In the present study, we further examined the underlying mechanism involved. Since insulin-induced insulin receptor substrate-1-associated phosphatidylinositol (PI) 3-kinase activity was not altered by Br-C4, we determined and found insulin activation of protein kinase B (PKB) and protein kinase Clambda (PKClambda) were both inhibited. However, time-course studies showed that only the inhibition of PKB activation correlated with the inhibition of insulin-stimulated glucose transport. In concert, insulin-stimulated Ser(473/474) phosphorylation on PKB(alpha/beta) were similarly decreased by Br-C4. The finding that okadaic acid-stimulated glucose transport and PKClambda activity were both inhibited by Br-C4 suggested that the effect of Br-C4 on Ser(473/474) phosphorylation was not mediated by protein phosphatase 2A. Moreover, whereas Br-C4 nearly abolished insulin-stimulated integrin-linked kinase (ILK) activity, it only inhibited insulin-stimulated PKB activity by 20%, implying that ILK was not the major kinase for Ser(473/474) phosphorylation. Taken together, these results support the notion that PKB is involved in insulin-stimulated glucose transport. In addition, Br-C4 seems to inhibit insulin-stimulated glucose transport via inhibiting insulin activation of PKB, probably by interfering with insulin activation of an upstream kinase responsible for the phosphorylation of Ser(473/474) residue.     

Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi-intact cells.

The vectorial transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis and medial Golgi compartments has been reconstituted using semi-intact (perforated) cells. The transport of VSV-G protein between successive compartments is measured by the sequential processing of the two N-linked oligosaccharide chains present on VSV-G protein to the endoglycosidase (endo) H-resistant structures which have unique electrophoretic mobilities during sodium dodecyl sulfate-gel electrophoresis. The appearance of a form of VSV-G which contains only one endo H-resistant oligosaccharide chain (GH1) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (GD), the latter being a processing reaction diagnostic of transport from the ER to the cis Golgi compartment. These results provide evidence that the cis Golgi compartment may contain in addition to alpha-1,2-mannosidase I, both N-acetylglueosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (GH2) after a second wave of vesicular transport. Processing of GH1 to GH2 in vitro occurs only after a lag period following the appearance of GH1; processing is sensitive to N-ethylmaleimide, guanosine-5'-O-(3-thiotriphosphate), and a synthetic peptide homologous to the rab1 protein effector domain, and processing is inhibited in the absence of free Ca2+ (in the presence of EGTA), reagents which potently inhibit ER to cis Golgi transport. These results suggest that VSV-G protein proceeds through at least two rounds of vesicular transport from the ER to the medial Golgi compartment for processing to the GH2 form, providing a model system to study the regulation of the vectorial membrane fission and fusion events involved in vesicular trafficking and organelle dynamics in the early stages of the secretory pathway.     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca(2+)-transients in human platelets. Role of a cdc2-related protein kinase.

The addition of either okadaic acid or calyculin A desensitizes human platelets to thrombin. One objective of this study was to determine which step(s) leading to secretion reactions may be affected by these protein phosphatase inhibitors. In a dose-dependent manner, okadaic acid or calyculin A inhibits phosphatidylinositol metabolism and Ca(2+)-transients. In all cases, calyculin A was approximately 10-fold more potent than okadaic acid, and it had maximal effects at a concentration of 1 microM. Although thrombin-induced rises in [Ca2+]i were diminished, an increase in the phosphorylation state of myosin light chains (MLC) was still observed. Changes in this phosphorylation were diminished, however, following the addition of thrombin to calyculin A-treated platelets that were loaded with dimethyl-BAPTA. These data demonstrate that calyculin A and okadaic acid lower agonist-induced Ca(2+)-transients, which in turn prevents responses such as secretion reactions. Calyculin A/okadaic acid-induced phosphorylation events were not diminished in BAPTA-loaded platelets, suggesting that these phosphorylations are Ca(2+)-insensitive. Thus, a second objective of this study was to identify the protein kinase(s) that was(were) responsible for the calyculin A-induced phosphorylations. In a platelet lysate system, calyculin A caused an increase in the incorporation of [32P]phosphate into p50. This phosphorylation event was identical to that observed in the intact platelet and was not mimicked by cAMP, cGMP, Ca2+, or a Ca2+/phospholipid/diacylglycerol mixture. Kinase activity was removed after the lysate was incubated with p13suc1-Sepharose. This suggests that a p13suc1-sensitive protein kinase, e.g., a cell cycle-dependent protein kinase, is responsible for the calyculin A-sensitive phosphorylation events.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hypertonic and sodium-free medium on transport of a membrane glycoprotein along the secretory pathway in cultured mammalian cells

Incubation of cultured cells in hypertonic medium and sodium-free medium have been shown to block transport at two different stages along the endocytic pathway. To determine the effects of these treatments on the exocytic pathway, we studied the transport of the membrane glycoprotein of vesicular stomatitis virus (VSV-G) in cells infected with tsO45 mutant virus. This mutant synthesizes a VSV-G that accumulates in the endoplasmic reticulum (ER) when cells are incubated at 39.5 degrees C. In addition, VSV-G accumulates in the post-ER pre-Golgi compartment when cells are incubated at 15 degrees C and in the trans-Golgi network (TGN) when cells are incubated at 18 degrees C. Upon transfer of cells to 32 degrees C in control medium, VSV-G exits each of these compartments and is transported to the cell surface. Incubation in sodium-free medium at 32 degrees C did not block transport from any of these three compartments. In contrast, incubation in hypertonic medium blocked export from the ER, transport from the pre-Golgi compartment to the Golgi complex, and transport from the TGN to the cell surface. Our results, in combination with previous studies, suggest that hypertonic medium blocks at least five distinct transport steps; the three exocytic steps described here, endocytosis from the cell surface, and transport of cell surface proteins into the Golgi complex. This raises the possibility that vesicular transport in different parts of the cell shares common elements that are inhibited by this treatment.