Ca(2+) sequestering in the early-branching amitochondriate protozoan Tritrichomonas foetus: an important role of the Golgi complex and its Ca(2+)-ATPase

Total membrane vesicles isolated from Tritrichomonas foetus showed an ATP-dependent Ca(2+) uptake, which was not sensitive to 10 microM protonophore FCCP but was blocked by orthovanadate, the inhibitor of P-type ATPases (I(50)=130 microM), and by the Ca(2+)/H(+) exchanger, A-23187. The Ca(2+) uptake was prevented also by thapsigargin, an inhibitor of the SERCA Ca(2+)-ATPases. The sensitivity of the Ca(2+) uptake by the protozoan membrane vesicles to thapsigargin was similar to that of Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. Fractionation of the total membrane vesicles in sucrose density gradient revealed a considerable peak of Ca(2+) transport activity that co-migrated with the Golgi marker guanosine diphosphatase (GDPase). Electron microscopy confirmed that membrane fractions of the peak were enriched with the Golgi membranes. The Golgi Ca(2+)-ATPase contributed to the Ca(2+) uptake by all membrane vesicles 80-85%. We conclude that: (i) the Golgi and/or Golgi-like vesicles form the main Ca(2+) store compartment in T. foetus; (ii) Ca(2+) ATPase is responsible for the Ca(2+) sequestering in this protozoan, while Ca(2+)/H(+) antiporter is not involved in the process; (iii) the Golgi pump of this ancient eukaryotic microorganism appears to be similar to the enzymes of the SERCA family by its sensitivity to thapsigargin.     

Effects of Brefeldin A on disassembly of the Golgi body in MDCK cells subjected to a Ca2+ shift at low temperature

When Madin-Darby canine kidney (MDCK) cells were grown in low-Ca2+ medium (LCM) the trans-Golgi cisternae, like those of cells maintained in high-Ca2+ medium (HCM), showed discrete localization of reaction product after thiamine pyrophosphatase (TPPase) staining. After exposure to Brefeldin A (BFA, 5 microg/ml) in LCM at 19 degrees C, the Golgi body dispersed and reaction product was distributed to the nuclear envelope and endoplasmic reticulum. The Golgi body reassembled in cells shifted back to HCM at 37 degrees C, with or without BFA, suggesting that low temperature and LCM exert synergistic effects in aiding dispersal of the Golgi apparatus in the presence of BFA. However, these results appear to be more directly correlated with the lack of defined cell polarity. Cells in LCM are unpolarized and both the centrosomes and the Golgi body are sub-nuclear in position, in contrast to their location in HCM where both organelles lie above the nucleus. The effects of BFA on the disassembly of the Golgi body therefore suggest that MDCK cells grown in LCM at low temperature cells are comparable to those non-polarized cell lines that are sensitive to BFA.     

Uncoupled ATP hydrolysis and thermogenic activity of the sarcoplasmic reticulum Ca2+-ATPase: coupling effects of dimethyl sulfoxide and low temperature

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the energy derived from ATP hydrolysis. During catalysis, part of the energy is used to translocate Ca(2+) across the membrane, and part is dissipated as heat. At 35 degrees C the heat released during the hydrolysis of each ATP molecule varies depending on the formation of a Ca(2+) gradient across the membrane. With leaky vesicles (no gradient) the heat released varies between 9 and 12 kcal/mol of ATP cleaved, and with intact vesicles (gradient), the heat released increases to 20-24 kcal/mol of ATP. After Ca(2+) accumulation, 82% of the Ca(2+)-ATPase activity is not coupled to Ca(2+) transport, and the ratio between Ca(2+) transported and ATP cleaved is 0.3. The addition of 20% dimethyl sulfoxide (v/v) to the medium or decreasing the temperature from 35 to 20 degrees C abolishes the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient. This is accompanied by a simultaneous inhibition of the uncoupled ATPase activity and an increase of the Ca(2+)/ATP ratio from 0.3 to 1.3-1.4. It is concluded that the uncoupled Ca(2+)-ATPase is responsible for both the low Ca(2+)/ATP ratio measured during transport and the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient.     

RNA Synthesis in Yersinia pestis During Growth Restriction in Calcium-Deficient Medium

Yersinia pestis requires 2.5 mM Ca(2+) for growth at 37 degrees C but not at 26 degrees C. After a shift from 26 to 37 degrees C in a Ca(2+)-deficient medium, an ordered series of metabolic alterations occur which result in transition from a growing cell to a viable but non-proliferating cell. The earliest known alteration in normal metabolism associated with this transition is a termination of net RNA synthesis. Competitive RNA/DNA hybridizations with uniformly labeled RNA and stable RNA competitor indicated identical mRNA to stable RNA ratios in growing cells and non-proliferating Ca(2+)-deprived cells. Similar hybridizations with pulse-labeled RNA demonstrated that growing cells synthesized 57% mRNA, 37% rRNA, and 5% tRNA, whereas Ca(2+)-deprived cells synthesized 95% mRNA, 4.7% rRNA, and 0.7% tRNA. After addition of radioactive uracil and rifampin to growing and Ca(2+)-deprived cells, decay of approximately 40 and 90% of the newly synthesized RNA was found for growing and Ca(2+)-deprived cells, respectively. The half-life of the mRNA was found to be 1.5 min for growing cells and 4.5 min for Ca(2+)-deprived cells. Y. pestis elicited increases in the levels of guanosine tetraphosphate and guanosine pentaphosphate in response to amino acid deprivation and yielded transient increases in the levels of these phosphorylated nucleotides after a shift from 26 to 37 degrees C. These increases were independent of Ca(2+) availability and preceded the alteration in RNA synthesis by more than 1 h. The levels of these phosphorylated nucleotides then stabilized at about 80 and 40 pmol for Ca(2+)-deprived and Ca(2+)-supplemented cultures, respectively, and did not increase further in the Ca(2+)-deprived culture at the time corresponding to the reduction in stable RNA synthesis. These findings indicate that the early lesion in RNA synthesis associated with the growth restriction of Ca(2+)-deprived Y. pestis reflects a block in stable RNA synthesis and that this effect is not mediated by guanosine tetraphosphate or guanosine pentaphosphate.     

Identification of the 16 degrees C compartment of the endoplasmic reticulum in rat liver and cultured hamster kidney cells

In many systems transfer between the endoplasmic reticulum and the Golgi apparatus is blocked at temperatures below 16 degrees C. In virus-infected cells in culture, a special membrane compartment is seen to accumulate. Our studies with rat liver show a similar response to temperature both in situ with slices and in vitro with isolated transitional endoplasmic reticulum fractions. With isolated transitional endoplasmic reticulum fractions, when incubated in the presence of nucleoside triphosphate and a cytosol fraction, temperature dependent formation of vesicles occurred with a Q10 of approximately 2 but was apparent only at temperatures greater than 12 degrees C. A similar response was seen in situ at 12 degrees C and 16 degrees C where fusion of transition vesicles with cis Golgi apparatus, but not their formation, was blocked and transition vesicles accumulated in large numbers. At 18 degrees C and below and especially at 8 degrees C and 12 degrees C, the cells responded by accumulating smooth tubular transitional membranes near the cis Golgi apparatus face. With cells and tissue slices at 20 degrees C neither transition vesicles nor the smooth tubular elements accumulated. Those transition vesicles which formed at 37 degrees C were of a greater diameter than those formed at 4 degrees C both in situ and in vitro. The findings show parallel responses between the temperature dependency of transition vesicle formation in vitro and in situ and suggest that a subpopulation of the transitional endoplasmic reticulum may be morphologically and functionally homologous to the 16 degrees C compartment observed in virally-infected cell lines grown at low temperatures.     

Transport of Ca2+ by diltiazem across the lipid bilayer in model liposomes.

The calcium channel antagonist diltiazem was examined for its ability to translocate Ca2+ from an aqueous medium to the nonpolar lipid milieu. We monitored the spectral changes caused by the drug-mediated cation transport at 37 degrees C in unilamellar vesicles made of dimyristoyl phosphatidylcholine (DMPC) and containing the calcium-sensitive dye arsenazo III trapped inside. Vesicle leakage or membrane fusion caused by diltiazem was assessed by the use of vesicles containing fluorescent indicators. These effects were, however, found to be insignificant compared with ion transport. The transport was negligible at temperatures below the liquid crystalline to gel transition temperature of DMPC indicating a carrier mechanism of ion transport. A quantitative analysis of the transport kinetics indicated that a 1:2 Ca(2+)-drug complex is formed inside the lipid. The calcium ionophoretic ability of diltiazem, combined with other related data, suggests a possible role for Ca2+ in the conformation of the drug in the lipid membrane milieu and in its interaction with the calcium channel.     

Hypothermia increases the gain of excitation-contraction coupling in guinea pig ventricular myocytes

Components of excitation-contraction (EC)-coupling were compared at 37 degrees C and 22 degrees C to determine whether hypothermia altered the gain of EC coupling in guinea pig ventricular myocytes. Ca(2+) concentration (fura-2) and cell shortening (edge detector) were measured simultaneously. Hypothermia increased fractional shortening (8.3 +/- 1.7 vs. 2.6 +/- 0.3% at 37 degrees C), Ca(2+) transients (157 +/- 33 vs. 35 +/- 5 nM at 37 degrees C), and diastolic Ca(2+) (100 +/- 9 vs. 60 +/- 6 nM at 37 degrees C) in field-stimulated myocytes (2 Hz). In experiments with high-resistance microelectrodes, the increase in contractions and Ca(2+) transients was accompanied by a twofold increase in action potential duration (APD). When voltage-clamp steps eliminated changes in APD, cooling still increased contractions and Ca(2+) transients. Hypothermia increased sarcoplasmic reticulum (SR) Ca(2+) stores (83 +/- 17 at 37 degrees C to 212 +/- 50 nM, assessed with caffeine) and increased fractional SR Ca(2+) release twofold. In contrast, peak Ca(2+) current was much smaller at 22 degrees C than at 37 degrees C (1.3 +/- 0.4 and 3.5 +/- 0.7 pA/pF, respectively). In cells dialyzed with sodium-free pipette solutions to inhibit Ca(2+) influx via reverse-mode Na(+)/Ca(2+) exchange, hypothermia still increased contractions, Ca(2+) transients, SR stores, and fractional release but decreased the amplitude of Ca(2+) current. The rate of SR Ca(2+) release per unit Ca(2+) current, a measure of EC-coupling gain, was increased sixfold by hypothermia. This increase in gain occurred regardless of whether cells were dialyzed with sodium-free solutions. Thus an increase in EC-coupling gain contributes importantly to positive inotropic effects of hypothermia in the heart.     

A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor

A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.     

Intracellular transport of influenza virus hemagglutinin to the apical surface of Madin-Darby canine kidney cells

The intracellular pathway followed by the influenza virus hemagglutinin (HA) to the apical surface of Madin-Darby canine kidney cells was studied by radioimmunoassay, immunofluorescence, and immunoelectron microscopy. To synchronize the migration, we used a temperature- sensitive mutant of influenza WSN, ts61, which, at the nonpermissive temperature, 39.5 degrees C, exhibits a defect in the HA that prevents its exit from the endoplasmic reticulum. Upon transfer to permissive temperature, 32 degrees C, the HA appeared in the Golgi apparatus after 10 min, and on the apical surface after 30-40 min. In the presence of cycloheximide, the expression was not inhibited, indicating that the ts defect is reversible; a wave of HA migrated to the cell surface, where it accumulated with a half time of 60 min. After passage through the Golgi apparatus the HA was detected in a population of smooth vesicles, about twice the size of coated vesicles, located in the apical half of the cytoplasm. These HA-containing vesicles did not react with anti- clathrin antibodies. Monensin (10 microM) delayed the surface appearance of HA by 2 h, but not the transport to the Golgi apparatus. Incubation at 20 degrees C retarded the migration to the Golgi apparatus by approximately 30 min and blocked the surface appearance by acting at a late stage in the intracellular pathway, presumably at the level of the post-Golgi vesicles. The initial appearance of HA on the apical surface was in the center; no preference was observed for the tight-junctional regions.     

Calcium depletion blocks proteolytic cleavages of plasma protein precursors which occur at the Golgi and/or trans-Golgi network. Possible involvement of Ca(2+)-dependent Golgi endoproteases.

The effects of calcium depletion on the proteolytic cleavage and secretion of plasma protein precursors were investigated in primary cultured rat hepatocytes and HepG2 cells. When the cells were incubated with A23187, the calcium-specific ionophore, in a medium lacking CaCl2, precursors of serum albumin and the third and fourth components of complement, C3 and C4, respectively, were found to be released into the medium. The addition of ionomycin or EGTA to the medium inhibited the processing of pro-C3 as well. Blocking the secretory pathway either at the mixed endoplasmic reticulum/Golgi in the presence of brefeldin A or at the endoplasmic reticulum/tubular-vesicular structure at a reduced temperature caused accumulation of pro-C3 within hepatocytes or HepG2 cells, indicating that the cleavage of the precursor occurs at a later stage of the secretory pathway. Once the blockade was released by incubating the cells either in the brefeldin A-free medium or at 37 degrees C, the secretion of plasma proteins resumed, irrespective of the presence of A23187. However, the processing of pro-C3 was almost completely inhibited in the presence of A23187, with only the precursor being released into the medium, implying that a decline in Ca2+ levels within the cell modulates the activity of a Golgi endoprotease responsible for the cleavage of pro-C3. When incubated with isolated Golgi membranes, pro-C3 secreted from Ca(2+)-depleted cells was cleaved in vitro into their subunits in the presence of Ca2+ but not in its absence, pointing to the involvement of a Ca(2+)-dependent Golgi endoprotease in the processing of pro-C3. These results collectively suggest that calcium depletion blocks the proteolytic cleavages of plasma protein precursors presumably by exhausting a Ca2+ pool available to the Ca(2+)-dependent processing enzyme(s) located at the Golgi and/or trans-Golgi network.     

Control of Intracellular Movement of Connexins by E-Cadherin in Murine Skin Papilloma Cells

The gap junctional intercellular communication-deficient mouse skin papilloma cell line P3/22 expresses Cx43 but not E-cadherin. The E-cadherin gene-transfected cells (P3E1) communicate in a calcium-dependent manner and they were used to study how E-cadherin restores the function of connexins. At low calcium, Cx43 molecules remain in the cytoplasm of P3E1 cells and appear at cell-cell contact areas only in high-calcium medium. While Cx43 is unphosphorylated in P3E1 cells in low-calcium medium, two phosphorylated bands appeared at high calcium. However, when Cx26, which has no C-terminal tail that can undergo phosphorylation, was expressed in P3E1 cells, this connexin also moved to the plasma membrane after the calcium shift and partly colocalized with Cx43, suggesting that C-terminal phosphorylation is not essential for E-cadherin-mediated intracellular transport of connexins. In low calcium, both Cx26 and Cx43 remained and colocalized in the endoplasmic reticulum. As early as 30 min after the shift to high-calcium medium, both Cx43 and Cx26 began to accumulate in the Golgi apparatus. Intracellular movement of connexins to the cytoplasmic membrane at high calcium was effectively blocked by cytochalasin D and brefeldin A. These results suggest that E-cadherin junction formation at high calcium leads to formation of actin cables, which directly or indirectly transport connexins from the cytoplasm to the cell-cell contact membranes via the Golgi apparatus.     

Intracellular vesicles involved in the transport of Semliki Forest virus membrane proteins to the cell surface.

The route of transport of Semliki Forest virus (SFV) membrane glycoproteins to the plasma membrane was studied using immunoperoxidase electron microscopy. SFV glycoproteins were localized in cultured BHK-21 fibroblasts infected with a temperature-sensitive mutant ts-1 of SFV, which shows a temperature-dependent, reversible defect in the transport of membrane glycoproteins to the cell surface. At 39 degrees C (restrictive temperature) the viral proteins were retained in the endoplasmic reticulum and the nuclear membrane. After shift of the infected cultures to 28 degrees C (permissive temperature) the proteins were synchronously transported to the Golgi complex. In the Golgi complex the labeled proteins were first (at 2.5 min) detected in large Golgi-associated vacuoles (GAV). Subsequently, i.e., at 5-30 min, the viral glycoproteins appeared in the cisternal stack: at 5 min the label was found in one or two of the proximal cisternae whereas at 15 or 30 min also the more distal cisternae were partially or uniformly labeled. At all time points examined after the temperature-shift, peroxidase label was found in 50 nm vesicles which were frequently coated. At 30 min, in addition to the 50 nm vesicles, larger 80 nm vesicles, which often had a cytoplasmic coat were labeled in the Golgi region. These results identify two major size classes of both coated and smooth vesicles which appear to function in the transport of the viral membrane proteins from the endoplasmic reticulum via distinct GAV and the stacked Golgi cisternae to the plasma membrane.     

Sequential intermediates in the transport of protein between the endoplasmic reticulum and the Golgi

Semi-intact cells, a cell population in which the plasma membrane is perforated to expose intact intracellular organelles (Beckers, C. J. M., Keller, D. S., and Balch, W. E. (1987) Cell 50, 523-534), efficiently reconstitute vesicular trafficking of protein from the endoplasmic reticulum (ER) to the cis Golgi compartment. We now extend these studies to biochemically dissect transport of protein between the ER and the Golgi into a series of sequential intermediate steps involved in the budding and fusion of carrier vesicles. At least two broad categories of transport intermediates can be detected, those that involve early steps in transport and those involved in late, fusion-related events. Early transport steps require the transport of protein through a novel intermediate compartment in which protein accumulates at reduced temperature (15 degrees C). We demonstrate that both entry and exit from this 15 degrees C compartment can be successfully reconstituted in vitro. A late step in delivery of protein to the cis Golgi compartment requires Ca2+ (pCa7) and is coincident with a step which is sensitive to a peptide analog which blocks interaction between the Rab family of small GTP-binding proteins and a downstream effector protein(s) (Plutner, H., Schwaninger, R., Pind, S., and Balch, W. E. (1990) EMBO J. 9, 2375-2384). The combined results suggest that a single round of vesicular transport between the ER and the Golgi involves a rapid transit through N-ethylmaleimide-sensitive, guanosine 5'-(3-O-thio)triphosphate-sensitive, ATP- and cytosol-dependent step(s) involved in vesicle formation or transport to a novel intermediate compartment, followed by a regulated fusion event triggered in the presence of Ca2+ and functional components interacting with member(s) of the Rab gene family.     

Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.     

LcrG, a secreted protein involved in negative regulation of the low-calcium response in Yersinia pestis.

The purpose of this study was to define the function of LcrG, the product of the first gene in the lcrGVHyopBD operon of the low-Ca(2+)-response (LCR) virulence plasmid of Yersinia pestis. We created a Y. pestis strain having an in-frame deletion in lcrG. This nonpolar mutant had an abnormal LCR growth phenotype: it was unable to grow at 37 degrees C in the presence of 2.5 mM Ca2+ ("Ca2+ blind") but was able to grow at 37 degrees C when 18 mM ATP was present. At 37 degrees C it failed to downregulate the expression and secretion of its truncated product (LcrG), V antigen, and YopM. All of these mutant properties were complemented by plasmids carrying normal lcrG. However, a nonpolar lcrE mutation and an lcrH mutation (both also causing a Ca(2+)-blind phenotype) were not complemented in this way. The Y. pestis parent strain expressed LcrG at 37 degrees C in the presence and absence of Ca2+ and transported it to the medium when Ca2+ was absent. We identified two LCR-regulated loci, lcrD and yscDEF, required for this transport. Complementation analysis of the Y. pestis lcrR strain previously shown to lack the expression of LcrG showed that the loss of LcrG but not of LcrR caused the Ca(2+)-blind phenotype of that mutant. Taken together, the results show that LcrG is a negative regulator of the LCR, perhaps functioning in Ca2+ sensing along with LcrE.     

Calcium adenosine triphosphatase and secretion of koilin membrane in the gizzard of the fowl.

The localization of calcium adenosine triphosphatase (Ca(2+)-ATPase) was determined histo- and ultracytochemically in the gizzard gland cells of the adult domestic fowl. Surface and chief gland cells exhibited faint and inconstant basolateral activity in contrast to basal cells, whose basolateral cell membrane constantly showed deposition on the external side. Intracellular enzyme activity was localized on the luminal aspect of Golgi membranes in all types of gland cells. Lysosomes also reacted positive for Ca(2+)-ATPase. Neither membranes of secretory vesicles nor cortical cytoplasm of the secretory pole exhibited enzyme activity. From these results it is speculated that calcium is not essentially involved in the secretion of the koilin membrane in terms of storage of the secretory material, transport to the secretory surface and release into the lumen. Ca(2+)-ATPase activity rather seems to be related to differentiation and maturation processes and to intracellular storage of Ca2+.     

Nucleocytoplasmic traffic of proteins.

We have used the synchronized formation of a mixed cytoplasm upon heterokaryon formation as a model for investigating the cisternal-specific transport of resident proteins between neighboring Golgi apparatus. Rat NRK and hamster 15B cells were fused by UV-inactivated Sindbis virus and then incubated for various time periods in the presence of cycloheximide. The resident Golgi apparatus proteins, rat GIMPc and Golgp 125, were localized with species-specific monoclonal antibodies. Immunofluorescent colocalization of rat and hamster Golgi membrane proteins was observed with a t1/2 of 1.75 h at 37 degrees C. Colocalization of resident, but not transient, Golgi membrane protein was concomitant with formation of a large extended Golgi complex and was accompanied by the acquisition of endoglycosidase H resistance by preexisting Golgp 125. Dispersal of the extended Golgi complex by nocodazole revealed that colocalization of resident Golgi proteins was due to intermixing of proteins in the same Golgi element rather than overlapping of closely apposed Golgi structures. Incubation of the polykaryons at 20 degrees C inhibited both the colocalization of GIMPc and Golgp 125 and the formation of an extended Golgi complex. Little change in the number of cisternae/stack in cross sections of the Golgi apparatus was observed upon cell fusion, and in the extended Golgi complex the hamster resident protein remained localized to one side of the Golgi stack. Surprisingly, the morphological identity of the rat and hamster Golgi units appeared to be maintained in the heterokaryons. These results suggest that the intermixing of resident Golgi membrane proteins requires direct physical continuity between Golgi elements and that resident Golgi membrane proteins are preferentially excluded from the non-clathrin-coated transport vesicles budding from Golgi cisternae.     

Endocytosis of mannose-6-phosphate binding sites by mouse T-lymphoma cells

The endocytosis and intracellular transport of mannose-6-phosphate conjugated to bovine serum albumin (Man-6-P:BSA) by mouse T-lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man-6-P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man-6-P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent- or 125I-labeled Man-6-P:BSA at 0 degree C revealed a uniform distribution of the Man-6-P binding sites over the cell surface. Competition experiments indicate that the Man-6-P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37 degrees C, endocytosis of Man-6-P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5-15-min incubation of cells at 37 degrees C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man-6-P:BSA in the Golgi region after 15-30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man-6-P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30-min incubation at 37 degrees C, the internalized Man-6-P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co-stained for acid phosphatase. These results demonstrate a temporal participation of clathrin-containing coated vesicles during the initial endocytosis of Man-6-P binding sites and that one step in the Man-6-P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.     

ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.     

Lipid mixing is mediated by the hydrophobic surfactant protein SP-B but not by SP-C.

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air/fluid interface of the lung. The excimer/monomer ratio of pyrene-labeled PC fluorescence intensities was used to investigate the capacity of the hydrophobic surfactant proteins, SP-B and SP-C, to induce lipid mixing between protein-containing small unilamellar vesicles and pyrene-PC-labeled small unilamellar vesicles. At 37 degrees C SP-B induced lipid mixing between protein-containing vesicles and pyrene-PC-labeled vesicles. In the presence of negatively charged phospholipids (PG or PI) the SP-B-induced lipid mixing was enhanced, and dependent on the presence of (divalent) cations. The extent of lipid mixing was maximal at a protein concentration of 0.2 mol%. SP-C was not capable of inducing lipid mixing at 37 degrees C not even at protein concentrations of 1 mol%. The SP-B-induced lipid mixing may occur during the Ca(2+)-dependent transformation of lamellar bodies into tubular myelin and the subsequent formation of the phospholipid monolayer.