多肽:N-乙酰氨基半乳糖转移酶2在SGC7901细胞中同时定位于高尔基体顺面囊和反面囊

多肽:N.乙酰氨基半乳糖转移酶(ppGalNAcT)在高尔基体中催化粘蛋白型O-糖基化的第一步.首先进行了人ppGalNAcT2多克隆抗体的制备和鉴定,进一步通过对分离的亚细胞结构进行蛋白质印迹分析,免疫细胞化学后共聚焦显微镜观察此抗体和两个高尔基体标记GS28(顺面高尔基体的分子标志)和TGN38(反面高尔基体的分子标志)来研究ppGalNAcT2在SGC7901细胞株中的亚细胞定位.结果表明:约有60%的ppGalNAcT2信号和Gs28共定位,大约36%的ppGalNAcT2信号和TGN38共定位.约有34%的TGN38和ppGalNAcT2信号重叠,而约38%的反面高尔基体标志和ppGalNAcT2重叠.结论是:在SGC7901中,ppGalNAcT2同时定位于高尔基体顺面囊和反面囊中,实验证实了在高尔基体中进行粘蛋白型O-糖基化的起始反应.     

Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.     

Localization of microsomal triglyceride transfer protein in the Golgi: possible role in the assembly of chylomicrons

Although a critical role of microsomal transfer protein (MTP) has been recognized in the assembly of nascent apolipoprotein B (apoB)-containing lipoproteins, it remains unclear where and how MTP transfers lipids in the secretory pathway during the maturational process of apoB lipidation. The aims of this study were to determine whether MTP functions in the secretory pathway as well as in the endoplasmic reticulum and whether its large 97-kDa subunit interacts with the small 58-kDa protein disulfide isomerase (PDI) subunit and apoB, particularly in the Golgi apparatus. Using a high resolution immunogold approach combined with specific polyclonal antibodies, the large and small subunits of MTP were observed over the rough endoplasmic reticulum and the Golgi. Double immunocytochemical detection unraveled the colocalization of MTP and PDI as well as MTP and apoB in these same subcellular compartments. To confirm the spatial contact of these proteins, Golgi fractions were isolated, homogenized, and incubated with an anti-MTP large subunit antibody. Immunoprecipitates were applied on SDS-PAGE and then transferred on to nitrocellulose. Immunoblotting the membrane with PDI and apoB antibodies confirmed the colocalization of these proteins with MTP. Furthermore, MTP activity assay disclosed a substantial triglyceride transfer in the Golgi fractions. The occurrence of membrane-associated apoB in the Golgi, coupled with its interaction with active MTP, suggests an important role for the Golgi in the biogenesis of apoB-containing lipoproteins.     

Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006;-1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006;-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [(3)H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006;-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.     

Identification of a novel isoform of microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) has been studied extensively, primarily because of its role in the assembly of very low density lipoproteins by the liver and chylomicrons by the intestine. Recent studies have suggested that MTP may also play key roles in other cellular processes. In this paper we report the identification of a novel splice variant of MTP in mice. This isoform, MTP-B, has a unique first exon located approximately 2.7 kilobases upstream of canonical MTP (MTP-A) exon 1. The alternative exon encodes 35 amino acids compared with 20 amino acids encoded by exon 1 of MTP-A. MTP-B represents approximately 90% of total MTP mRNA in mouse adipocytes and 3T3-L1 cells and <5% in mouse liver and intestine. Expression of the alternate isoform in mouse liver was confirmed by mass spectrometry. Co-transfection of COS cells with truncated forms of apoB and either MTP-A or MTP-B demonstrated that both isoforms are effective in the assembly and secretion of nascent apoB-containing lipoproteins. Confocal microscopy of 3T3-L1 cells transfected with enhanced green fluorescent protein or DsRed fusions of the two proteins revealed that MTP-A is localized to the endoplasmic reticulum, whereas MTP-B localizes primarily to the Golgi complex in these cells. We conclude that MTP-B functions similarly to MTP-A in lipoprotein assembly. However, in nonlipoprotein-secreting cells, such as the adipocyte, MTP-B may have different localization properties, perhaps reflecting a distinct role in lipid storage and mobilization.     

Apolipoprotein B-related gene expression and ultrastructural characteristics of lipoprotein secretion in mouse yolk sac during embryonic development.

In mice, the yolk sac appears to play a crucial role in nourishing the developing embryo, especially during embryonic days (E) 7;-10. Lipoprotein synthesis and secretion may be essential for this function: embryos lacking apolipoprotein (apo) B or microsomal triglyceride transfer protein (MTP), both of which participate in the assembly of triglyceride-rich lipoproteins, are apparently defective in their ability to export lipoproteins from yolk sac endoderm cells and die during mid-gestation. We therefore analyzed the embryonic expression of apoB, MTP, and alpha-tocopherol transfer protein (alpha-TTP), which have been associated with the assembly and secretion of apoB-containing lipoproteins in the adult liver, at different developmental time points. MTP expression or activity was found in the yolk sac and fetal liver, and low levels of activity were detected in E18.5 placentas. alpha-TTP mRNA and protein were detectable in the fetal liver, but not in the yolk sac or placenta. Ultrastructural analysis of yolk sac visceral endoderm cells demonstrated nascent VLDL within the luminal spaces of the rough endoplasmic reticulum and Golgi apparatus at E7.5 and E8.5. The particles were reduced in diameter at E13.5 and reduced in number at E18.5;-19.The data support the hypothesis that the yolk sac plays a vital role in providing lipids and lipid-soluble nutrients to embryos during the early phases (E7;-10) of mouse development. secretion in mouse yolk sac during embryonic development.     

GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus

The subcellular localization, interacting partners, and function of GS15, a Golgi SNARE, remain to be established. In our present study, it is revealed that unlike proteins (Bet1 and the KDEL receptor) cycling between the Golgi and the intermediate compartment (IC, inclusive of the ER exit sites), GS15 is not redistributed into the IC upon incubation at 15 degrees C or when cells are treated with brefeldin A. Immuno-electron microscopy (immuno-EM) reveals that GS15 is mainly found in the medial-cisternae of the Golgi apparatus and adjacent tubulo-vesicular elements. Coimmunoprecipitation experiments suggest that GS15 exists in a distinct SNARE complex that contains SNAREs (syntaxin5, GS28, and Ykt6) that are implicated in both ER-to-Golgi and intra-Golgi transport but not with SNAREs involved exclusively in ER-to-Golgi traffic. Furthermore, components of COPI coat can be selectively coimmunoprecipitated with GS15 from Golgi extracts. Overexpression of mutant forms of GS15 affects the normal distribution of cis- and medial-Golgi proteins (GS28, syntaxin 5, and Golgi mannosidase II), whereas proteins of the trans-Golgi and TGN (Vti1-rp2/Vti1a and syntaxin 6) and Golgi matrix/scaffold (GM130 and p115) are less affected. When the level of GS15 is reduced by duplex 21-nt small interfering RNA (siRNA)-mediated knockdown approach, diverse markers of the Golgi apparatus are redistributed into small dotty and diffuse labeling, suggesting an essential role of GS15 in the Golgi apparatus.     

Oxysterol binding protein-dependent activation of sphingomyelin synthesis in the golgi apparatus requires phosphatidylinositol 4-kinase IIα

Cholesterol and sphingomyelin (SM) associate in raft domains and are metabolically coregulated. One aspect of coordinate regulation occurs in the Golgi apparatus where oxysterol binding protein (OSBP) mediates sterol-dependent activation of ceramide transport protein (CERT) activity and SM synthesis. Because CERT transfer activity is dependent on its phosphatidylinositol 4 phosphate [PtdIns(4)P]-specific pleckstrin homology domain, we investigated whether OSBP activation of CERT involved a Golgi-associated PtdIns 4-kinase (PI4K). Cell fractionation experiments revealed that Golgi/endosome-enriched membranes from 25-hydroxycholesterol-treated Chinese hamster ovary cells had increased activity of a sterol-sensitive PI4K that was blocked by small interfering RNA silencing of OSBP. Consistent with this sterol-requirement, OSBP silencing also reduced the cholesterol content of endosome/trans-Golgi network (TGN) fractions containing PI4KIIα. PI4KIIα, but not PI4KIIIβ, was required for oxysterol-activation of SM synthesis and recruitment of CERT to the Golgi apparatus. However, neither PI4KIIα nor PI4KIIIβ expression was required for 25-hydroxycholesterol-dependent translocation of OSBP to the Golgi apparatus. The presence of OSBP, CERT, and PI4KIIα in the TGN of oxysterol-stimulated cells suggests that OSBP couples sterol binding or transfer activity with regulation of PI4KIIα activity, leading to CERT recruitment to the TGN and increased SM synthesis.     

Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network

An in vitro transport assay, established with a modified Shiga toxin B subunit (STxB) as a marker, has proved to be useful for the study of transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN). Here, we modified this assay to test antibodies to all known soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that have been shown to localize in the Golgi and found that syntaxin 5, GS28, Ykt6, and GS15 antibodies specifically inhibited STxB transport. Because syntaxin 5, GS28, Ykt6, and GS15 exist as a unique SNARE complex, our observation indicates that these four SNAREs function as a complex in EE/RE-TGN transport. The importance of GS15 in EE/RE-TGN transport was further demonstrated by a block in recombinant STxB transport in HeLa cells when GS15 expression was knocked down by its small interfering iRNA. Morphological analyses showed that some GS15 and Ykt6 were redistributed from the Golgi to the endosomes when the recycling endosome was perturbed by SNX3-overexpression, suggesting that GS15 and Ykt6 might cycle between the endosomes and the Golgi apparatus. Further studies indicated that syntaxin 5 and syntaxin 16 exerted their role in EE/RE-TGN transport in an additive manner. The kinetics of inhibition exhibited by syntaxin 16 and syntaxin 5 antibodies is similar.     

Identify Golgi protein types with modified Mahalanobis discriminant algorithm and pseudo amino acid composition

The Golgi apparatus is an important eukaryotic organelle. Successful prediction of Golgi protein types can provide valuable information for elucidating protein functions involved in various biological processes. In this work, a method is proposed by combining a special mode of pseudo amino acid composition (increment of diversity) with the modified Mahalanobis discriminant for predicting Golgi protein types. The benchmark dataset used to train the predictor thus formed contains 95 Golgi proteins in which none of proteins included has ≥40% pairwise sequence identity to any other. The accuracy obtained by the jackknife test was 74.7%, with the ROC curve of 0.772 in identifying cis-Golgi proteins and trans-Golgi proteins. Subsequently, the method was extended to discriminate cis-Golgi network proteins from cis-Golgi network membrane proteins and trans-Golgi network proteins from trans-Golgi network membrane proteins, respectively. The accuracies thus obtained were 76.1% and 83.7%, respectively. These results indicate that our method may become a useful tool in the relevant areas. As a user-friendly web-server, the predictor is freely accessible at http://immunet.cn/SubGolgi/.     

Compartmentation of the Golgi complex: brefeldin-A distinguishes trans- Golgi cisternae from the trans-Golgi network

The Golgi complex is composed of at least four distinct compartments, termed the cis-, medial, and trans-Golgi cisternae and the trans-Golgi network (TGN). It has recently been reported that the organization of the Golgi complex is disrupted in cells treated with the fungal metabolite, brefeldin-A. Under these conditions, it was shown that resident enzymes of the cis-, medial, and trans-Golgi return to the ER. We report here that 300-kD mannose 6-phosphate receptors, when pulse-labeled within the ER of brefeldin-A-treated cells, acquired numerous N-linked galactose residues with a half time of approximately 2 h, as measured by their ability to bind to RCA-I lectin affinity columns. In contrast, Limax flavus lectin chromatography revealed that less than 10% of these receptors acquired sialic acid after 8 h in brefeldin-A. Two lines of evidence suggested that proteins within and beyond the TGN did not return to the ER in the presence of brefeldin-A. First, the majority of 300-kD mannose 6-phosphate receptors present in the TGN and endosomes did not return to the ER after up to 6 h in brefeldin-A, as determined by their failure to contact galactosyltransferase that had relocated there. Moreover, although mannose 6-phosphate receptors did not acquire sialic acid when present in the ER of brefeldin-A-treated cells, they were readily sialylated when labeled at the cell surface and transported to the TGN. These experiments indicate that galactosyltransferase, a trans-Golgi enzyme, returns to the endoplasmic reticulum in the presence of brefeldin-A, while the bulk of sialyltransferase, a resident of the TGN, does not. Our findings support the proposal that the TGN is a distinct, fourth compartment of the Golgi apparatus that is insensitive to brefeldin-A.     

The calcium-binding protein p54/NEFA is a novel luminal resident of medial Golgi cisternae that traffics independently of mannosidase II

A new Golgi resident, p54, has been demonstrated in several eukaryotic species and in multiple organs. Based on Triton X-114 partition, carbonate extraction and trypsin protection assays, p54 behaved as an extrinsic membrane protein, facing the luminal compartment. p54 was purified by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry as NEFA, a calcium-binding protein (Barnikol-Watanabe et al., 1994, Biol. Chem. Hoppe Seyler, 375, 497-512). By immunofluorescence, p54/NEFA essentially colocalized with the medial Golgi marker mannosidase II, and did not overlap with the cis-Golgi marker p58, nor with the trans-Golgi network (TGN) marker TGN38. By immuno-electron microscopy, p54/NEFA localized in the medial cisternae and in Golgi-associated vesicles. p54/NEFA remained associated with mannosidase II despite Golgi disruption by nocodazole, caffeine, or, to some extent, potassium depletion (a new procedure to induce Golgi disassembly), but the two markers rapidly dissociated upon brefeldin A treatment and at metaphase, and reassociated upon drug removal and at the end of anaphase. Since p54/NEFA is a peripheral luminal membrane constituent, its distinct trafficking from the transmembrane marker mannosidase II suggests a novel Golgi retention mechanism, by strong association of this soluble protein with another integral transmembrane resident.     

Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells

Return of cell surface glycoproteins to compartments of the secretory pathway has been examined in HepG2 cells comparing return to the trans- Golgi network (TGN), the trans/medial- and cis-Golgi. Transport to these sites was studied by example of the transferrin receptor (TfR) and the serine peptidase dipeptidylpeptidase IV (DPPIV) after labeling these proteins with the N-hydroxysulfosuccinimide ester of biotin on the cell surface. This experimental design allowed to distinguish between glycoproteins that return to these biosynthetic compartments from the cell surface and newly synthesized glycoproteins that pass these compartments during biosynthesis en route to the surface. Reentry to the TGN was measured in that surface glycoproteins were desialylated with neuraminidase and were monitored for resialylation during recycling. Return to the trans-Golgi was traced measuring the transfer of [3H]fucose residues to recycling surface proteins by fucosyltransferases. To study return to the cis-Golgi, surface proteins were metabolically labeled in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM). As a result surface proteins retained N-glycans of the oligomannosidic type. Return to the site of mannosidase I in the medial/cis-Golgi was measured monitoring conversion of these glycans to those of the complex type after washout of dMM. Our data demonstrate that DPPIV does return from the cell surface not only to the TGN, but also to the trans-Golgi thus linking the endocytic to the secretory pathway. In contrast, no reentry to sites of mannosidase I could be detected indicating that the early secretory pathway is not or is only at insignificant rates accessible to recycling DPPIV. In contrast to DPPIV, TfR was very efficiently sorted from endosomes to the cell surface and did not return to the TGN or to other biosynthetic compartments in detectable amounts, indicating that individual surface proteins are subject to different sorting mechanisms or sorting efficiencies during recycling.     

Acquisition of triacylglycerol transfer activity by microsomal triglyceride transfer protein during evolution

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of neutral-lipid-rich apolipoprotein B (apoB) lipoproteins. Previously we reported that the Drosophila MTP transfers phospholipids but does not transfer triglycerides. In contrast, human MTP transfers both lipids. To explore the acquisition of triglyceride transfer activity by MTP, we evaluated amino acid sequences, protein structures, and the biochemical and cellular properties of various MTP orthologues obtained from species that diverged during evolution. All MTP orthologues shared similar secondary and tertiary structures, associated with protein disulfide isomerase, localized to the endoplasmic reticulum, and supported apoB secretion. While vertebrate MTPs transferred triglyceride, invertebrate MTPs lacked this activity. Thus, triglyceride transfer activity was acquired during the transition from invertebrates to vertebrates. Within vertebrates, fish, amphibians, and birds displayed 27%, 40%, and 100% triglyceride transfer activity compared to mammals. We conclude that MTP triglyceride transfer activity first appeared in fish and speculate that the acquisition of triglyceride transfer activity by MTP provided for a significant advantage in the evolution of larger and more complex organisms.     

Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly

The microsomal triglyceride transfer protein (MTP) is necessary for the proper assembly of the apolipoprotein B containing lipoproteins, very low density lipoprotein and chylomicrons. Recent research has significantly advanced our understanding of the role of MTP in these pathways at the molecular and cellular level. Biochemical studies suggest that initiation of lipidation of the nascent apolipoprotein B polypeptide may occur through a direct association with MTP. This early lipidation may be required to allow the nascent polypeptide to fold properly and therefore avoid ubiquitination and degradation. Concerning the addition of core neutral lipids in the later stages of lipoprotein assembly, cell culture studies show that MTP lipid transfer activity is not required for this to occur for apolipoprotein B-100 containing lipoproteins. Likewise, MTP does not appear to directly mediate addition of core neutral lipid to nascent apoB-48 particles. However, new data indicate that MTP is required to produce triglyceride rich droplets in the smooth endoplasmic reticulum which may supply the core lipids for conversion of nascent, dense apoB-48 particles to mature VLDL. In addition, assembly of dense apolipoprotein B-48 containing lipoproteins has been observed in mouse liver in the absence of MTP. As a result of these new data, an updated model for the role of MTP in lipoprotein assembly is proposed.     

GRIP domain-mediated targeting of two new coiled-coil proteins,GCC88 and GCC185, to subcompartments of the trans-Golgi network

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the trans-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of HeLa cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the trans-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.     

Fractionation of Primary Cultured Cerebellar Neurons: Distribution of Sialyltransferases Involved in Ganglioside Biosynthesis

Primary cultured neurons were fractionated using sucrose density gradients. The activities of four sialyltransferases (GM3, GD3, GD1a, and GT1a synthase) involved in ganglioside biosynthesis were assayed in the collected fractions. The distribution of GM3 synthase coincided with that of mannosidase II, an enzyme assumed to be a cis-Golgi marker. Both enzymes were mainly associated with the more dense fraction. GD1a and GT1a synthase activities, on the other hand, were mainly recovered in the less dense fraction. Moreover, they were colocalized with thiamine pyrophosphatase, an enzyme assumed to be a marker of the late Golgi (trans-Golgi and trans-Golgi network). GD3 synthase activity was equally distributed between both fractions. These results are integrated in a model of ganglioside biosynthesis.     

Human placenta secretes apolipoprotein B-100-containing lipoproteins

Supply of lipids from the mother is essential for fetal growth and development. In mice, disruption of yolk sac cell secretion of apolipoprotein (apo) B-containing lipoproteins results in embryonic lethality. In humans, the yolk sac is vestigial. Nutritional functions are instead established very early during pregnancy in the placenta. To examine whether the human placenta produces lipoproteins, we examined apoB and microsomal triglyceride transfer protein (MTP) mRNA expression in placental biopsies. ApoB and MTP are mandatory for assembly and secretion of apoB-containing lipoproteins. Both genes were expressed in placenta and microsomal extracts from human placenta contained triglyceride transfer activity, indicating expression of bioactive MTP. To detect lipoprotein secretion, biopsies from term placentas were placed in medium with [(35)S]methionine and [(35)S]cysteine for 3-24 h. Upon sucrose gradient ultracentrifugation of the labeled medium, fractions were analyzed by apoB-immunoprecipitation. (35)S-labeled apoB-100 was recovered in d approximately 1.02-1.04 g/ml particles (i.e. similar to the density of plasma low density lipoproteins). Electron microscopy of negatively stained lipoproteins secreted from placental tissue showed spherical particles with a diameter of 47 +/- 10 nm. These results demonstrate that human placenta expresses both apoB and MTP and consequently synthesize and secrete apoB-100-containing lipoproteins. Placental lipoprotein formation constitutes a novel pathway of lipid transfer from the mother to the developing fetus.     

Apolipoprotein B-containing lipoprotein assembly in microsomal triglyceride transfer protein-deficient McA-RH7777 cells

Microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein (apo) B-containing lipoproteins. Previously, we demonstrated that the N-terminal 1,000 residues of apoB (apoB:1000) are necessary for the initiation of apoB-containing lipoprotein assembly in rat hepatoma McA-RH7777 cells and that these particles are phospholipid (PL) rich. To determine if the PL transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB:1000-containing lipoproteins, we employed microRNA-based short hairpin RNAs (miR-shRNAs) to silence Mttp gene expression in parental and apoB:1000-expressing McA-RH7777 cells. This approach led to 98% reduction in MTP protein levels in both cell types. Metabolic labeling studies demonstrated a drastic 90–95% decrease in the secretion of rat endogenous apoB100-containing lipoproteins in MTP-deficient McA-RH7777 cells compared with cells transfected with negative control miR-shRNA. A similar reduction was observed in the secretion of rat endogenous apoB48 under the experimental conditions employed. In contrast, MTP absence had no significant effect on the synthesis, lipidation, and secretion of human apoB:1000-containing particles. These results provide strong evidence in support of the concept that in McA-RH7777 cells, acquisition of PL by apoB:1000 and initiation of apoB-containing lipoprotein assembly, a process distinct from the conventional first-step assembly of HDL-sized apoB-containing particles, do not require MTP. This study indicates that, in hepatocytes, a factor(s) other than MTP mediates the formation of the PL-rich primordial apoB:1000-containing initiation complex.     

Profilin I attached to the Golgi is required for the formation of constitutive transport vesicles at the trans-Golgi network

Profilin I was identified, by mass spectrometric sequencing and immunoblotting, as a component of purified Golgi cisternae from HepG2 cells. Binding to the Golgi was verified by indirect immunofluorescence in MT-1 cells showing that a fraction of profilin I colocalizes with TGN38, a marker of the trans-Golgi network (TGN). Studying the formation of constitutive exocytic vesicles at the TGN in a cell-free system demonstrated that cytosolic profilin I has no effect, while incubation of Golgi cisternae with a profilin I-specific antibody reduced vesicle formation by about 50%. Notably, the antibody displaces a fraction of the Golgi-bound dynamin II indicating that profilin I may indirectly promote vesicle formation by supporting the binding of dynamin II to the Golgi membrane. The impact of dynamin II on vesicle formation is demonstrated by incubating the Golgi with the proline-rich domain of dynamin II which concomitantly displaces dynamin II and inhibits vesicle formation. The data provide evidence that profilin I attaches to the Golgi apparatus and is required for the formation of constitutive transport vesicles.