Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells

The relationship between apoB-100 and the membrane of the endoplasmic reticulum (ER) has been studied by a combination of pulse-chase methodology and subcellular fractionation. HepG2 cells were pulse-labeled with [35S]methionine for 3 min and chased with cold methionine for periods between 0 and 20 min. ApoB-100 and albumin, present in the membrane as well as in the luminal content of the ER vesicles, were isolated after each chase period. The results indicated that apoB-100 was cotranslationally bound to the membrane of the ER, and from this membrane-bound form, was transferred to the lumen after a delay of 10-15 min. Albumin was, as could be expected for a typical secretory protein, cotranslationally sequestered in the lumen of the ER. Apo-B-100-containing lipoproteins present in the microsomal lumen were analyzed by ultracentrifugation in a sucrose gradient. ApoB-100 occurred on rounded particles in three density regions: (i) d 1.1065-1.170 g/ml (Fraction I), (ii) d 1.011-1.045 g/ml (Fraction II), and (iii) d less than 1.011 g/ml (Fraction III). Fraction I, isolated from cells cultured in the absence of oleic acid, contained a homogenous population of particles with a mean diameter of approximately 200 A. Fraction I isolated from cells cultured in the presence of oleic acid was slightly more heterogeneous and had a mean diameter of approximately 250 A. Fractions II and III had mean diameters of 300 and 500 A, respectively. Cholesterol esters and triacylglycerol were the quantitatively dominating lipid constituents of all three fractions. Pulse-chase experiments indicated that Fraction I contained the newly assembled lipoproteins. With increasing chase time, the apoB-100 radioactivity was redistributed from Fraction I to Fractions II and III, indicating that Fraction I is converted into Fractions II and III during the intracellular transfer. Particles corresponding to Fractions II and III were by far the most abundant lipoproteins found in the medium. The results presented support the possibility of a sequential assembly of apoB-100-containing lipoproteins.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

Differential labeling of rat hepatic Golgi and serum very low density lipoprotein apoprotein B variants

The synthesis of apoB-100 and apoB-48 by rat liver was investigated by studying the apoB complement of very low density lipoproteins (VLDL) from hepatic perfusates and Golgi fractions. The relative amounts of apoB-100 and apoB-48 in perfusate and Golgi VLDL as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis were similar to those in serum VLDL. To investigate the relative rates of synthesis of the VLDL B proteins, rats were injected intraportally with tritiated amino acid, and hepatic Golgi and serum VLDL were isolated from 7.5 to 120 min later. In hepatic Golgi VLDL, apoB-100 and apoE were maximally labeled at 15 min after the tritiated amino acid pulse. In contrast, VLDL apoB-48 attained maximum radioactivity at 30 min after isotope injection. In serum VLDL, apoB-100 and apoE were maximally labeled at 30 min post-isotope injection, while activity in apoB-48 peaked at 60 min. The data suggest that the synthesis of the B proteins and incorporation into rat liver nascent VLDL are independently regulated. The differential labeling patterns of the VLDL B proteins may be explained by an intracellular pool of apoB-48 that is larger than that of apoB-100. An alternative explanation of the results is that apoB-100 is a precursor to apoB-48.     

Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment

The synthesis and secretion of apolipoprotein (apo) B-100 have been studied in a human hepatoblastoma cell line, the Hep G2 cells. Pulse-chase analysis showed that apoB-100 was not quantitatively recovered in the culture medium. To reveal the intracellular degradation of apoB-100 prior to secretion, cells were incubated with 1 microgram/ml Brefeldin A (BFA) which impeded protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus and the fate of apoB-100 retained in the cells was traced at 37 degrees C. A significant amount of intracellular apoB-100 (40-60%/h) was degraded during the chase period, whereas apoA-1 remained intact. ApoB-100 degradation was temperature dependent, no degradation was observed below 20 degrees C. This degradation process was not inhibited by chloroquine, leupeptin, pepstatin, and chymostatin, suggesting that lysosomal proteases were not involved and that apoB-100 was degraded in a pre-Golgi compartment which is either part of, or closely related to, the ER. Preincubation of cells with low density lipoproteins (LDL) induced a 22-32% increase in the degradation of apoB-100. This result raised the possibility that secretion of apoB-100 might be regulated through the intracellular degradation of apoB-100. These results suggest the existence of the degradation pathway for apoB-100 in a pre-Golgi compartment and an unique regulatory mechanism for apoB-100 secretion.     

The assembly and secretion of apoB 100 containing lipoproteins in Hep G2 cells. Evidence for different sites for protein synthesis and lipoprotein assembly

Pulse-chase studies combined with subcellular fractionation indicated that LpB 100 (i.e. the apoprotein B (apoB) 100 containing lipoproteins) was released to the lumen of the secretory pathway in a subcellular fraction enriched in smooth vesicles, and referred to as SMF (the smooth membrane fraction). The migration of SMF during gradient ultracentrifugation as well as kinetic studies indicated that the fraction was derived from a pre-Golgi compartment, probably the smooth endoplasmic reticulum (ER). Only small amounts of LpB 100 could be detected during these pulse-chase experiments in the subcellular fractions derived from the rough endoplasmatic reticulum (RER). SMF contained the major amount of the diacylglycerol acyltransferase activity present in the ER, while the major amount of membrane bound apoB 100 was present in the RER. Pulse-chase studies of the intracellular transfer of apoB 100 demonstrated the formation of a large membrane-bound preassembly pool in the ER, while no significant amount of apoB 100 radioactivity was present in the membrane of the Golgi apparatus. The maximal radioactivity of LpB 100, recovered from the ER or the Golgi lumen, was small compared with the radioactivity recovered from the ER membrane, indicating that the assembled LpB 100 rapidly leaves the cells. This in turn indicates that the rate-limiting step in the secretion of apoB 100 was the transfer of the protein from the ER membrane to the LpB 100 in the lumen. A portion of the intracellular pool of apoB 100 was not secreted but underwent posttranslational degradation.     

The role of leaders in intracellular transport and secretion of the insulin precursor in the yeast Saccharomyces cerevisiae.

Pulse-chase analysis of folded and misfolded insulin precursor (IP) expressed in Saccharomyces cerevisiae was performed to establish the requirements for intracellular transport and the influence of the secretory pathway quality control mechanisms on secretion. Metabolic labelling of the IP expressed in S. cerevisiae showed that the effect of a leader was to stabilise the IP in the endoplasmic reticulum (ER), and facilitate intracellular transport of the fusion protein and rapid secretion. The first metabolically labelled IP appeared in the culture supernatant within 2-4 min of chase, and most of the secreted IP appeared within the first 15 min of chase. After enzymatic removal of the leader in a late Golgi apparatus compartment, the IP followed one of two routes: (1) to the plasma membrane and hence to the culture supernatant, or (2) to a Golgi or post-Golgi compartment from which secretion was restricted. Combined secretion and intracellular retention of the IP reflected either saturation of a Golgi or post-Golgi compartment and secretion as a consequence of overexpression, or competition between secretion and intracellular retention. IP which was misfolded, either due to amino acid substitution or because disulphide bond formation had been prevented with dithiothreitol (DTT), was transported from the ER to the Golgi apparatus but then retained in a Golgi or post-Golgi compartment and not exported to the culture supernatant.     

Intracellular transport of the transmembrane glycoprotein G of vesicular stomatitis virus through the Golgi apparatus as visualized by electron microscope radioautography

The intracellular migration of G protein in vesicular stomatitis virus-infected cells was visualized by light and electron microscope radioautography after a 2-min pulse with [3H]mannose followed by nonradioactive chase for various intervals. The radioactivity initially (at 5-10 min) appeared predominantly in the endoplasmic reticulum, and the [3H]mannose-labeled G protein produced was sensitive to endoglycosidase H. Silver grains were subsequently (at 30-40 min) observed over the Golgi apparatus, and the [3H]mannose-labeled G protein became resistant to endoglycosidase H digestion. Our data directly demonstrate the intracellular transport of a plasmalemma-destined transmembrane glycoprotein through the Golgi apparatus.     

The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes.

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.     

Secretion kinetics of hydroxyproline-containing macromolecules in carrot root discs

Short-term pulse-chase studies using radioactive L-proline on carrot tissue support the classical endomembrane route for secretory proteins. Labelled hydroxyproline residues were first detectable in fractions containing the endoplasmic reticulum (ER) after a 5 min pulse. Upon chase-out this fraction looses and, initially, the Golgi apparatus (GA) fraction gains radioactivity. Unlike ER and GA fractions which show chase-out characteristics a plasma membrane (PM) containing-fraction reveals retention of some of the radioactivity.     

A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.

The Euglena precursor to the small subunit of ribulose-15-bisphosphate carboxylase/oxygenase (pSSU) is a polyprotein. To determine the transport route from cytoplasm to chloroplast, Euglena was pulse labeled with 35S-sulfate and the organelles were separated on sucrose gradients. After a pulse, pSSU was found in the endoplasmic reticulum (ER) and Golgi apparatus. During a chase, ER-and Golgi-localized pSSU decreased concomitant with the appearance of SSU in chloroplasts. SSU was not found in pSSU-containing ER and Golgi fractions. Na2CO3 did not remove pSSU from ER or Golgi membranes, indicating that it was an integral membrane protein. pSSU was inserted in vitro into canine microsomes, and Na2CO3 did not remove pSSU from the microsomal membrane. The in vivo and in vitro experiments show that Euglena pSSU is inserted into the ER membrane and transported as an integral membrane protein to the Golgi apparatus before chloroplast import and polyprotein processing.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

apoA-IV tagged with the ER retention signal KDEL perturbs the intracellular trafficking and secretion of apoB

To examine the role of apolipoprotein A-IV (apoA-IV) in the intracellular trafficking and secretion of apoB, COS cells were cotransfected with microsomal triglyceride transfer protein (MTP), apoB-41 (amino terminal 41% of apoB), and either native apoA-IV or apoA-IV modified with the carboxy-terminal endoplasmic reticulum (ER) retention signal, KDEL (apoA-IV-KDEL). As expected, apoA-IV-KDEL was inefficiently secreted relative to native apoA-IV. Coexpression of apoB-41 with apoA-IV-KDEL reduced the secretion of apoB-41 by approximately 80%. The apoA-IV-KDEL effect was specific, as neither KDEL-modified forms of human serum albumin or apoA-I affected apoB-41 secretion. Similar results were observed in McA-RH7777 rat hepatoma cells, which express endogenous MTP. The full inhibitory effect of apoA-IV-KDEL on apoB secretion was observed only for forms of apoB containing a minimum of the amino-terminal 25% of the protein (apoB-25). However, apoA-IV-KDEL inhibited the secretion of both lipid-associated and lipid-poor forms of apoB-25. Dual-label immunofluorescence microscopy of cells transfected with native apoA-IV and apoB-25 revealed that both apolipoproteins were localized to the ER and Golgi, as expected. However, when apoA-IV-KDEL was cotransfected with apoB-25, both proteins localized primarily to the ER. These data suggest that apoA-IV may physically interact with apoB in the secretory pathway, perhaps reflecting a role in modulating the process of triglyceride-rich lipoprotein assembly and secretion.     

Subcellular localization of the EGF receptor maturation process

The glycosylation and the processing of the epidermal growth factor (EGF) receptor are suggested to play a crucial role(s) in the activation of ligand binding activity. To examine whether the receptor acquires EGF binding activity in the endoplasmic reticulum (ER) or in the Golgi complex, we carried out parallel kinetic analysis of the EGF binding activity and the intracellular transport of the newly synthesized receptor by immunoprecipitation with the anti-EGF receptor antibody B4G7 using the EGF receptor hyperproducing cell line NA. The kinetic analysis revealed that a receptor capable of binding EGF appeared after 30 to 60 min labeling with [35S]methionine. Pulse-chase experiments also indicated that the receptor capable of binding EGF appeared after a 30-min pulse with a 30-min chase. Subcellular fractionation analysis indicated that the newly synthesized receptor was present in the Golgi complex after labeling with [35S]methionine for 30 min. After a 30-min chase, the Mr 170K receptor appeared in the Golgi complex and plasma membrane. Thus, these results together indicated that after a 30-min pulse incubation a fraction of the EGF receptors have been transported from the ER to the Golgi complex; however, the receptor is unable to bind EGF. Although the EGF receptor appeared on the cell surface after a 30-min pulse with a 30-min chase, only half of the receptors are capable of binding EGF. Therefore, the EGF receptor acquires ligand binding activity at a late stage of the maturation process, most likely in the Golgi complex.     

Fractionation of suspension cultures of wild carrot and kinetics of membrane labeling

Summary Wild carrot (Daucus carota L.) cells, grown in suspension culture, were labeled with radioactive precursors and fractionated into constituent membranes to be analyzed for specific radioactivity. Results show rapid incorporation of [³H] leucine into endoplasmic reticulum (ER)-, Golgi apparatus-, and plasma membrane/tonoplast-enriched fractions. The time lag between incorporation into ER and its appearance in Golgi apparatus or plasma membrane/tonoplast were less than 5 minutes. With an average time of 3–4 minutes for cisternal formation estimated from studies with monensin, and an average of 5 cisternae per dictyosome (total transit time of 15–20 minutes), it was not possible to account for early incorporation of radioactivity into plasma membranes by passage of proteins from ER to plasma membrane via the Golgi apparatus. To account for the findings, it would appear that at least some proteins were delivered to the plasma membrane via the first membranes that exited (i.e., mature face vesicles) from the Golgi apparatus post-pulse and that some of these proteins had been translated and inserted into membranes at or near the mature face of the Golgi apparatus.     

Apolipoprotein B-100: immunolocalization and synthesis in human intestinal mucosa

Despite the evidence that the human small intestine produces two separate species of apoB mRNA encoding for B-100 and B-48, there is a paucity of data concerning the expression of the latter form in this organ. Using a high resolution immunogold approach, with specific polyclonal antibodies and a panel of monoclonal antibodies (2D8, 3A10, 4G3), both forms of apoB (B-48 and B-100) were revealed over enterocytes of pediatric intestinal samples. Intense labeling was observed over microvilli, apical smooth membrane vesicles, multivesicular bodies, the basolateral membrane, as well as the trans Golgi region. Only low labeling was found over the rough endoplasmic reticulum (rER). Similar patterns of apoB distribution characterized both duodenal and jejunal regions. The presence of labeling over the Golgi apparatus and rER suggests a synthetic activity of both forms of apoB by the epithelial cells. To test this hypothesis, human intestine was incubated with [3H]leucine, homogenized, and subjected to immunoprecipitation for apoB. Immunoprecipitates contained radioactivity mainly in apoB-48 with relatively small amounts in apoB-100 when examined by NaDodSO4-polyacrylamide gel electrophoresis. These findings were further supported by the biochemical determination of apoB-100 and apoB-48 in chylomicron particles isolated from thoracic duct lymph of a human donor. Taken together, our data suggest that the human intestine is able to synthesize and to express the apoB-100.     

The conversion of apoB100 low density lipoprotein/high density lipoprotein particles to apoB100 very low density lipoproteins in response to oleic acid occurs in the endoplasmic reticulum and not in the Golgi in McA RH7777 cells

The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [35S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.     

Assembly of very low density lipoprotein in the hepatocyte. Differential transport of apoproteins through the secretory pathway

The transport of the apolipoprotein (apo) constituents of hepatic very low density lipoprotein (VLDL) through the secretory pathway was investigated with estrogen-induced chick hepatocytes in primary culture. Cell monolayers were pulse-labeled with [3H]leucine and, after differing periods of chase with unlabeled leucine, were subjected to subcellular fractionation for 3H-apoprotein analysis. The first-order rate constants for transit of apoB, apoA-I, and apoII through the endoplasmic reticulum (ER) and Golgi were estimated using a three-compartment (ER, Golgi, and extracellular medium) kinetic analysis. The results indicate that apoB resides in the ER (t1/2 = 26 min) for a shorter period of time than in the Golgi (t1/2 = 43 min). For apoII, the t1/2 for transport through the ER and Golgi are 43 and 49 min, respectively. ApoA-I transits the ER at a rate (t1/2 = 6 min) much faster than apoB, apoII, and virtually all other secretory proteins. Upon reaching the Golgi, the rate of movement of apoA-I is markedly reduced (t1/2 = 28 min). Thus, in contrast to current models of protein secretion, the rate-limiting step in the secretion of VLDL apoproteins from the cell is transport through the Golgi, not the ER. Examination of the steady-state distribution of the apoproteins in the ER and Golgi support this conclusion. To characterize the intracellular transport process further, the distribution of apoproteins between the lumenal contents of the ER and Golgi and the membranes which delineate these compartments was determined after steady-state labeling with [3H]leucine. Approximately 50% of the apoB in the ER and in a dense, early Golgi fraction was membrane-associated, whereas in a less dense or late Golgi compartment, only 20% was bound to membranes. ApoII was also associated with the membranes of the ER and Golgi to a significant extent. In contrast, apoA-I was primarily localized lumenally throughout the secretory pathway. The occurrence of membrane-associated apoproteins in the Golgi, coupled with their slow rate of transit through this compartment suggests a major role for the Golgi in the assembly of the constituents of VLDL, and suggests that interaction of apoproteins (apoB) with the membranes of the Golgi is required for the maturation of VLDL.     

Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells

Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatment-induced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulse-chase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.     

Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and golgi apparatus with [3H]leucine

Summary Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [³H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.