Lipid transport to avian oocytes and to the developing embryo.

Studies of receptor-mediated lipoprotein metabolic pathways in avian species have revealed that physiological intricacies of specific cell types are highly analogous to those in mammals. A prime example for the power of comparative studies across different animal kingdoms, elucidated in the chicken, is that the expression of different lipoprotein receptors in somatic cells and oocytes are the key to oocyte growth. In avian species, yolk precursor transport from the hen's liver to rapidly growing oocytes and the subsequent transfer of yolk nutrients via the yolk sac to the developing embryo are highly efficient processes.

Reversal of antifungal resistance mediated by ABC efflux pumps from Candida albicans functionally expressed in yeast.

The enhanced efflux of antifungal drugs through ATP-binding cassette (ABC) transporters constitutes a major cause of clinical multidrug resistance (MDR). The inhibition of drug efflux pumps by specific compounds is considered to be a feasible strategy to overcome clinical antifungal resistance. Therefore, several blockers of mammalian and yeast ABC drug pumps, including FK506, propafenones, as well as the antifungal drug terbinafine were tested for their capacity to reverse CDR-mediated azole resistance in bakers yeast and in clinical isolates of Candida albicans.

Regulation by estrogen of synthesis and secretion of apolipoprotein A-I in the chicken hepatoma cell line, LMH-2A.

The synthesis and secretion of apolipoprotein A-I (apoA-I) in response to the treatment with estrogen were investigated in the chicken hepatoma cell line, LMH-2A. Exposure of these cells to exogenous estrogen for up to 48 h results in a decrease of apoA-I production, as evident from Western blotting, immunoprecipitation, and immunofluorescence experiments. Likewise, the secretion of apoA-I is also decreased in estrogen-treated cells when compared to controls.

Molecular characterization of the first avian LDL receptor: role in sterol metabolism of ovarian follicular cells.

Low levels of expression and sluggish sterol-mediated regulation have been likely reasons for the failure to molecularly characterize a bona fide LDL receptor (LDLR) in egg-laying species to date. The overall structure of the chicken LDLR, delineated here by cDNA cloning, has been conserved in evolution, since hallmark properties of mammalian LDLRs are already present in the avian protein. The chicken receptor appears to prefer LDL over VLDL as ligand, in compliance with its main role in providing lipoprotein-derived cholesterol for steroid production in ovarian follicular cells.

The low-density lipoprotein receptor family: genetics, function, and evolution.

With ever increasing sophistication in molecular biological approaches, the low-density lipoprotein receptor supergene family continues to grow rapidly. From the well-defined key role of these receptors in lipoprotein metabolism, the new members move the field into many different and diverse physiologic and developmental areas. We observe an expansion of the functional spectrum of the family members, which is due to 1) the binding to their extracellular domains of more and more components lacking homology to apolipoproteins, and 2) the recently uncovered interaction of the receptors' cytoplasmic tails with adaptor proteins that are part of signaling pathways.

From cholesterol transport to signal transduction: low density lipoprotein receptor, very low density lipoprotein receptor, and apolipoprotein E receptor-2.

The discovery of an ever growing number of low density lipoprotein (LDL) receptor gene family members has triggered research into many different directions. Here we first summarize the results of classical studies on the role of the LDL receptor in cholesterol transport, the structure/function relationships delineated with the help of LDL receptor mutations in familial hypercholesterolemia, and the elegant way in which cells regulate cholesterol at the transcriptional level. The second part deals with a multifunctional, structurally very close relative, the very low density lipoprotein (VLDL) receptor.

The major chicken egg envelope protein ZP1 is different from ZPB and is synthesized in the liver.

The extracellular matrix surrounding vertebrate oocytes is called the zona pellucida in mammals and perivitelline membrane (pvm) in birds. We have analyzed this structure in chicken follicles and laid eggs and have identified a 95-kDa component of the pvm, which, by protein sequencing, shows homology to mammalian zona pellucida proteins. Surprisingly, we could not detect this protein in ovarian granulosa cells or oocytes but instead found high levels in the liver of the laying hen.

Degradation of a short-lived glycoprotein from the lumen of the endoplasmic reticulum: the role of N-linked glycans and the unfolded protein response.

We are studying endoplasmic reticulum-associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI(332), containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI(332) was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of approximately 45 min was followed by a second phase of threefold accelerated degradation.

Multiple involvement of clusterin in chicken ovarian follicle development. Binding to two oocyte-specific members of the low density lipoprotein receptor gene family.

The interaction of the female germ cell with somatic cells during the development of the ovarian follicle in the chicken provides a prime system to study gene expression. Here, we have uncovered the involvement of clusterin, the function(s) of which is still poorly understood, in this complex process. As revealed by molecular cloning, chicken clusterin is a 428-residue protein that migrates at 70 kDa on SDS-polyacrylamide gel electrophoresis and possesses most of the structural features of its mammalian successors.

Oocyte growth in the chicken: receptors and more.

The laying hen's left ovary (the ovary on the right side regresses during embryonic development) at any given time contains 5-8 follicles with vitellogenic oocytes, i.e., female germ cells of increasing size ranging from 8 to approx.

The chicken homologue of zona pellucida protein-3 is synthesized by granulosa cells.

Oocyte development within avian ovarian follicles is an intricate process involving yolk deposition and the formation of extraoocytic matrices. Of these, the perivitelline membrane (pvm) not only plays a role in sperm binding but also provides mechanical support for the large oocyte's journey through the oviduct after ovulation. To date we have focused on the mechanisms for uptake of yolk precursors into oocytes of the chicken; now we extend our studies to a detailed analysis of the pvm.

Expression and conservation of apolipoprotein AIV in an avian species.

In birds, intestinally derived lipoproteins are thought to be secreted directly into the portal vein rather than to enter the circulation via the lymphatic system as in mammals. Hepatic clearance of these so-called portomicrons must be rapid, but the protein(s) mediating their catabolism, presumably analogues of the 36-kDa mammalian apolipoprotein E, have not been identified. In searching for such a mediator(s), we have isolated a hitherto unknown 38-kDa protein from chicken serum, which we identified by microsequencing and molecular cloning as a counterpart to mammalian apolipoprotein AIV (apoAIV).

Molecular cloning and expression of an avian rab5 homolog.

A chicken rab5 cDNA was isolated that contains the complete open reading frame for a protein of 216 amino acids, which, by comparison with available rab5 sequences from other species, is most closely related to the rab5c isoform. Two rab5 transcripts of 1.3 and 1.

Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome.

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded.

Receptor-associated protein in an oviparous species is correlated with the expression of a receptor variant.

The biosynthesis of proteins containing cysteine-rich domains requires chaperones for their correct folding. For instance, the 39-kDa receptor-associated protein (RAP) aides in the cell-surface targeting of newly synthesized members of the mammalian low density lipoprotein receptor (LDLR) gene family, which contains tandemly arranged clusters of hexacysteine repeats. In the chicken, an LDLR relative with eight such repeats is expressed as two different splice variant forms in cell type-specific fashion (Bujo, H.

Novel members of the low density lipoprotein receptor superfamily and their potential roles in lipid metabolism.

The list of LDL receptor superfamily relatives is still growing. The two most recently discovered family branches are (1) a set of receptors characterized by expression in brain and a very close relationship to the LDL and VLDL receptors, and (2) highly cross-species (man, mouse, rabbit, chicken) conserved complex mosaic receptors which contain structural domains so far not found in the superfamily. At present, we know very little about the physiological function(s) of these molecules.

Functional protein prenylation is required for the brefeldin A-dependent retrograde transport from the Golgi apparatus to the endoplasmic reticulum.

In cells exposed to brefeldin A (BFA), enzymes of the Golgi apparatus are redistributed to the endoplasmic reticulum (ER) by retrograde membrane flow, where they may cause modifications on resident ER proteins. We have used a truncated form of the rough ER-specific type I transmembrane glycoprotein ribophorin I as a probe to detect Golgi glycosyltransferases relocated to the ER in a BFA-dependent fashion. This polypeptide (RI332) comprises the 332 amino-terminal amino acids of ribophorin I and behaves like a luminal ER protein when expressed in HeLa cells.

Estrogen dependence of synthesis and secretion of apolipoprotein B-containing lipoproteins in the chicken hepatoma cell line, LMH-2A.

The chicken hepatoma cell line LMH-2A, which permanently overexpresses the chicken estrogen receptor, was used to study the synthesis and secretion of lipoproteins in response to treatment with estrogen. In the absence of the hormone, only small amounts of apolipoprotein B (apoB) and no apolipoprotein VLDL II (apoII) were found in cell extracts. After treatment of cells with moxestrol, a stable estrogen derivative, for 24 to 48 h, a dramatic increase in the quantities of these lipoproteins was observed both in cell extracts and in the medium.

The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole.

We have recently demonstrated that the Pdr5 ATP binding cassette multidrug transporter is a short-lived protein, whose biogenesis involves cell surface targeting followed by endocytosis and delivery to the vacuole for proteolytic turnover [Egner, R., Mahé, Y., Pandjaitan, R.

Chicken lecithin-cholesterol acyltransferase. Molecular characterization reveals unusual structure and expression pattern.

Rapidly growing oocytes in the laying hen are, in addition to the liver, targets of the so-called "reverse cholesterol transport" (RCT) (Vieira, P.M., Vieira, A.

Retinol in avian oogenesis: molecular properties of the carrier protein.

Normal embryo development in oviparous (egg-laying) species requires the coordinated targeting to growing oocytes of nutrients and regulatory molecules such as retinol, the precursor of active retinoids. Serum retinol-binding protein (RBP) is the major carrier protein for retinol in the circulatory system of vertebrates. In oviparous animals, RBP is thought to function in the delivery of retinol to yolk, in analogy to other important nutrients and vitamins known to accumulate within the oocyte.

The chicken oocyte receptor for lipoprotein deposition recognizes alpha 2-macroglobulin.

alpha 2-Macroglobulin (alpha 2M), a major plasma component in all vertebrates, is proposed to function as a broad spectrum protease inhibitor. The alpha 2M-proteinase complex (activated alpha 2M; alpha 2M*) is removed rapidly by receptor-mediated endocytosis in the liver. Here we demonstrate by Western blotting that alpha 2M is also present in the yolk of chicken oocytes.

Chicken yolk contains bona fide high density lipoprotein particles.

Lipoproteins, the major nutrient source for developing embryos in egg-laying species, are thought to be transported from the circulation of the hen to the yolk of growing oocytes. In order to fully understand the contribution of the different lipoprotein species to oocyte growth, yolk formation, and embryo development, we have started to elucidate the relationships between the high density lipoproteins (HDL) in serum with the hitherto uncharacterized yolk HDL fraction. Immunoblotting with antibodies against apolipoprotein (apo) A-I, the major protein moiety of circulating HDL, revealed, for the first time, significant amounts of this protein in yolk.

Secretion of peptides and proteins lacking hydrophobic signal sequences: the role of adenosine triphosphate-driven membrane translocators.

In this review, we will emphasize the role of ATP-dependent membrane transporters in protein export and intracellular protein trafficking in prokaryotic and eukaryotic cells. ATP-binding-cassette (ABC)-transport proteins, also termed "traffic ATPases," belong to a superfamily of ubiquitous ATP-driven membrane transporters that share extensive sequence similarity and highly conserved domain organization. They are implicated in a remarkable variety of transmembrane transport processes, including the transport of ions, heavy metals, sugars, anticancer drugs, amino acids, oligopeptides, and proteins.