The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium.

The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine (Arfrp1(vil-/-)) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. Arfrp1(vil-/-) enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the Arfrp1(vil-/-) epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylo-microns and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells.

Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1.

The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sensitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1(ad)(-/-)) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1(ad)(-/-) adipocytes. In addition, in Arfrp1(ad)(-/-) adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4.

Substantial changes of cellular iron homeostasis during megakaryocytic differentiation of K562 cells.

We investigated the remodeling of iron metabolism during megakaryocytic development of K562 cells. Differentiation was successfully verified by increase of the megakaryocytic marker CD61 and concomitant decrease of the erythroid marker γ-globin. The reduction of erythroid properties was accompanied by changes in the cellular iron content and in the expression of proteins regulating cellular iron homeostasis. Independent of available inorganic or transferrin-bound extracellular iron, total intracellular iron increases while the iron-to-protein ratio decreases. The iron exporter ferroportin is downregulated within 1-6 h, followed by downregulation of transferrin receptor-1 (TfR1) and ferritin heavy chain (H-ferritin) mainly after 24-48 h. The hemochromatosis protein-1, a ligand of TfR1, peaked after 24 h. All effects were independent of iron supply with the exception of H-ferritin, which was restored by excess iron. While alterations of CD61, TfR1 and ferritin expression were revoked by a protein kinase C inhibitor, downregulation of ferroportin remained unaffected.

The transferrin receptor-1 membrane stub undergoes intramembrane proteolysis by signal peptide peptidase-like 2b.

The successive events of shedding and regulated intramembrane proteolysis are known to comprise a fundamental biological process of type I and II membrane proteins (e.g. amyloid precursor protein, Notch receptor and pro-tumor necrosis factor-α). Some of the resulting fragments were shown to be involved in important intra- and extracellular signalling events. Although shedding of the human transferrin receptor-1 (TfR1) has been known for > 30 years and soluble TfR1 is an accepted diagnostic marker, the fate of the remaining N-terminal fragment (NTF) remains unknown. In the present study, we demonstrate for the first time that TfR1-NTF is subject to regulated intramembrane proteolysis and, using MALDI-TOF-TOF-MS, we have identified the cleavage site as being located C-terminal from Gly-84. We showed that the resulting C-terminal peptide is extracellularly released after regulated intramembrane proteolysis and it was detected as a monomer with an internal disulfide bridge. We further identified signal peptide peptidase-like 2a and mainly signal peptide peptidase-like 2b as being responsible for the intramembrane proteolysis of TfR1-NTF.

A lysine-free mutant of epidermal growth factor as targeting moiety of a targeted toxin.

AIMS: Elevated levels of epidermal growth factor (EGF) receptor are observed on several human tumors, e.g. cervical carcinoma and mamma carcinomas. The natural ligand EGF is an alternative to established antibodies and tyrosine kinase inhibitors for targeting EGF receptor-overexpressing tumor cells for therapy. Conjugations of compounds to EGF lack the necessary homogeneity for an intended application, since several amino acids may react with the chemical linker. MAIN METHODS: We designed an EGF variant (EGF(RR)) in which the two lysines were substituted with arginine (K28R and K48R). EGF(RR) was fused to the protein toxin saporin to obtain a model protein for detailed analyses on EGF receptor binding and on both the enzymatic activity of saporin and the cytotoxicity of the fusion protein. KEY FINDINGS: The mutation decreased the enzymatic activity of saporin 2.3-fold and the binding of EGF(RR) retained its specificity for EGF receptor while increasing the Kd 5.5-fold. In spite of these differences the cytotoxicity of the fusion protein was unchanged in comparison to a fusion protein with EGF both when applied alone and in combination with cytotoxicity augmenting saponin. SIGNIFICANCE: We conclude that EGF(RR) retained its ability to bind with high specificity to EGF receptor and is thus suitable for a number of chemical linkage applications such as targeting drugs or dyes to EGF receptor-expressing cells.

The ARF-like GTPase ARFRP1 is essential for lipid droplet growth and is involved in the regulation of lipolysis.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1(ad-/-)) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1(ad-/-) mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1(ad-/-) adipocytes, suggesting that lipid droplet growth is defective in Arfrp1(ad-/-) mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1(ad-/-) mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.

ADP-ribosylation factor-like GTPase ARFRP1 is required for trans-Golgi to plasma membrane trafficking of E-cadherin.

ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1(-/-) embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1(vil)(-/-)), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1(vil)(-/-) enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk(-)Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, and p120(ctn) from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function.

Knockout of Arfrp1 leads to disruption of ARF-like1 (ARL1) targeting to the trans-Golgi in mouse embryos and HeLa cells.

ADP-ribosylation factor related protein 1 (ARFRP1) is a member of the ARF-family of GTPases which operate as molecular switches in the regulation of intracellular protein traffic. Deletion of the mouse Arfrp1 gene leads to embryonic lethality during early gastrulation, suggesting that ARFRP1 is required for cell adhesion-related processes. Here we show that ARFRP1 specifically controls targeting of ARL1 and its effector Golgin-245 to the trans-Golgi. GTP-bound ARFRP1 (ARFRP1-Q79L mutant) is associated with Golgi membranes and co-localized with the GTPase ARL1. In contrast, the guanine nucleotide exchange defective ARFRP1 mutant (ARFRP1-T31N) clusters within the cytosol. ARFRP1-T31N or depletion of endogenous ARFRP1 by RNA interference disrupts the Golgi association of ARL1 and of the GRIP-domain protein Golgin-245 and alters the distribution of a trans-Golgi network marker, syntaxin 6. In contrast, the targeting of two other Golgi-associated proteins, GM130 and giantin, was unaffected. Furthermore, in Arfrp1-/ - embryos ARL1 dislocated from Golgi membranes whereas it was associated with intracellular membranes in wild-type embryos. These data suggest that lethality of Arfrp1 knockout embryos is due to a specific disruption of protein targeting, e.g., of ARL1 and Golgin-245, to the Golgi.

Characterization of the human SLC2A11 (GLUT11) gene: alternative promoter usage, function, expression, and subcellular distribution of three isoforms, and lack of mouse orthologue.

GLUT11 (SLC2A11) is a class II sugar transport facilitator which exhibits highest similarity with the fructose transporter GLUT5 (about 42%). Here we demonstrate that separate exons 1 (exon 1A, exon 1B, and exon 1C) of the SLC2A11 gene generate mRNAs of three GLUT11 variants (GLUT11-A, GLUT11-B, and GLUT11-C) that differ in the amino acid sequence of their N-termini. All three 5'-flanking regions of exon 1A, exon 1B and exon 1C exhibited promoter activity when expressed as luciferase fusion constructs in COS-7 cells. 5'-RACE-PCR, quantitative real-time PCR, and Northern blot analysis performed with specific probes for exon 1A, 1B and 1C demonstrated that GLUT11-A is expressed in heart, skeletal muscle, and kidney, GLUT11-B in kidney, adipose tissue, and placenta, and GLUT11-C in adipose tissue, heart, skeletal muscle, and pancreas. Surprisingly, mice and rats lack the SLC2A11 gene. When expressed in Xenopus oocytes, all three GLUT11 isoforms transport glucose and fructose but not galactose. There was no apparent difference in the subcellular distribution of the three isoforms expressed in COS-7 cells. Our data indicate that different promoters and splicing of the human SLC2A11 gene generate three GLUT11 isoforms which are expressed in a tissue specific manner but do not appear to differ in their functional characteristics.