Assembly and Secretion of Very Low Density Lipoproteins Containing Apolipoprotein B48 in Transfected McA-RH7777 Cells

Vukmirica, Jelena; Tran, Khai; Liang, Xiquan; Shan, Jing; Yuan, Jane; Miskie, Brooke A; Hegele, Robert A.; Resh, Marilyn D.; Yao, Zemin

doi:10.1074/jbc.m211995200

Cited by 17 publications

(16 citation statements)

References 30 publications

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“…Although such rapid shuttling of G proteins from Golgi to PM is unanticipated, it has been previously suggested based on the localization of palmitoyl lacking ␣ subunits that acylation at endomembranes facilitates PM localization of a G protein (29,30). The 2BP inhibition may also inhibit an unidentified process that is not related to palmitoylation of a G protein subunit or a binding protein (13,27).…”

Section: G Protein Shuttling Is Likely Diffusion-mediated-mentioning

confidence: 99%

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Shuttling of G Protein Subunits between the Plasma Membrane and Intracellular Membranes

Chisari

Saini

Kalyanaraman

et al. 2007

Journal of Biological Chemistry

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Heterotrimeric G proteins (␣␤␥) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1 ⁄ 2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.Heterotrimeric G proteins are known to function on the cytosolic surface of the plasma membrane (PM) 2 in response to the stimulation of transmembrane receptors by most of the extracellular signals a mammalian cell senses (1-4). The PM localization of a G protein is facilitated by individual lipid modifications of the ␣ and ␤␥ subunits (5). Consistent with a model of G protein action on the PM, activated G protein subunits have been shown to modulate the functions of effectors such as adenylyl cyclase, phospholipase C, and ion channels which are also PM-localized (1-4). However, the spatiotemporal dynamics of G protein localization in a living cell has not been examined to determine whether G protein subunits are stably constrained to the PM. Although there is longstanding evidence for the existence of G protein subunits in intracellular membranes such as the Golgi complex and suggestions that they affect protein trafficking (6 -11), it has been unclear as to how they reach endomembranes and if they are native to these membranes or in transit to the PM.Here we used a variety of imaging methods on live cells with fluorescent protein-tagged G protein subunits including photoswitchable Dronpa (12) to observe G protein movement. A fluorescence resonance energy transfer (FRET)-based G protein sensor was used to examine whether G proteins reside constitutively in the endomembranes. The results show that G protein subunits shuttle rapidly between the PM and endomembranes in cells in the basal state maintaining a pool of G proteins in the endomembranes. We show that the shuttling is likely diffusive and not vesicle-mediated. Furthermore, 2-bromopalmitate (2BP), an inhibitor of palmitoylation (13), inhibited shuttling, suggesting that it may be regulated by acylation. MATERIALS AND METHODS Details of chemicals, expression constructs, cell lines, transfection, and treatment of cells are as in Saini et al. (41). Dronpa fluorescent proteins were introduced downstream of Gly-92 in␣ o as in the ␣ o -CFP construct that we have shown previously to possess normal...

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Section: G Protein Shuttling Is Likely Diffusion-mediated-mentioning

confidence: 99%

“…Refs. 17 and 26), it is also known to inhibit other processes unrelated to palmitoylation (13,27). In FRAP experiments 30 min of treatment with 50 M 2BP inhibited the recovery of fluorescence in the Golgi region of cells expressing ␣ o -GFP, YFP-␥ 11 , or YFP-␥ 9 ( Fig.…”

Section: G Protein Shuttling Is Likely Diffusion-mediated-mentioning

confidence: 99%

Shuttling of G Protein Subunits between the Plasma Membrane and Intracellular Membranes

Chisari

Saini

Kalyanaraman

et al. 2007

Journal of Biological Chemistry

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“…Preparation of Expression Plasmids-The expression plasmids encoding human apoB-100 (39), B-48 (40), and B-17 (41) cDNA, respectively, were prepared as described previously. Numbers following apoB represent the percent of full-length apoB (apoB-100).…”

Section: Methodsmentioning

confidence: 99%

Missense Mutations in APOB within the βα1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia

Burnett

Zhong

Jiang

et al. 2007

Journal of Biological Chemistry

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Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n ‫؍‬ 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n ‫؍‬ 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the ␣-helical domain within the N terminus of apoB. Thus, proper folding of the ␣-helical domain of apoB-100 is essential for efficient secretion. Apolipoprotein (apo)6 B, a large amphipathic glycoprotein, plays a central role in human lipoprotein metabolism (1, 2). The human apoB gene (APOB) is located on chromosome 2 and produces, via a unique mRNA editing process (3), two forms of apoB, namely apoB-48 (2152 amino acids) and apoB-100 (4536 amino acids) (4, 5). ApoB-48 is the truncated form of apoB-100 consisting of the N-terminal 48% of full-length apoB-100. ApoB-48 is synthesized in the intestine and is essential for the formation and secretion of chylomicrons. ApoB-100 is synthesized in the liver and is an essential structural component of very low density lipoprotein (VLDL) and its metabolic products, intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), and is also a ligand for the LDL receptor. Unlike humans, the rat liver produces both apoB-100 and apoB-48, and both forms can assemble VLDL (6).A pentapartite model for human apoB-100 has been proposed, which depicts a five-domain structure composed of alternating amphipathic ␣-helices and amphipathic ␤-strands, namely ␤␣1-␤1-␣2-␤2-␣3 (7). The ␤␣1 domain is a mixture of 13 amphipathic ␤-strands and 17 amphipathic ␣-helices, whose amino acids share extensive sequence homologies to the yolk protein lipovitellin (7-9). The apoB ␤␣1 domain has been modeled on the structure of silver lamprey lipovitellin, in which the 13 ␤-strands (amino acids 21-263) form a ␤-barrel, whereas the 17 ␣-helices (amino acids 440 -592) form a two-layered helical bundle (10). There is an interface between the ␣-helical bundle * This work was supported by the Heart and Stroke Foundation of Ontario . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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“…HDL particles provide an excellent test system, because glycosylation, phosphorylation, truncation, signal peptide cleavages, pro-peptide cleavages, sulfation, fatty acylation, and alternative splicing are all established for apolipoproteins [16][17][18][19][20]. Acylation by palmitate or palmitoleate [18][19][20][21][22][23] should, in principle, be detectable on these gels, based on our previous virtual 2-D gel observations of E. coli proteolipids mulI and osmE (SwissProt accession numbers P02937 and P23933, respectively) [13].…”

Section: Virtual 2-d Gel Electrophoresismentioning

confidence: 99%

Virtual two‐dimensional gel electrophoresis of high‐density lipoproteins

Loo

Yam²,

Loo

et al. 2004

Electrophoresis

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Assembly and Secretion of Very Low Density Lipoproteins Containing Apolipoprotein B48 in Transfected McA-RH7777 Cells

Cited by 17 publications

References 30 publications

Shuttling of G Protein Subunits between the Plasma Membrane and Intracellular Membranes

Shuttling of G Protein Subunits between the Plasma Membrane and Intracellular Membranes

Missense Mutations in APOB within the βα1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia

Virtual two‐dimensional gel electrophoresis of high‐density lipoproteins

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