YFP Fusion Research Articles

Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase Fet3p from Saccharomyces cerevisiae.

The Fet3 protein in Saccharomyces cerevisiae is a multicopper oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe(2+) to Fe(3+); this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe(2+), one of which is essential also to the coupling of the ferroxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K(M) for Fe(2+) exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferroxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in (59)Fe uptake. For the Fet3p(E185D) mutant, K(M) for iron was 300-fold greater than the wild-type K(M), while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe(2+ )binding to Fet3p and to the subsequent trafficking of the Fe(3+) produced to Ftr1p.

Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants.

Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death may occur from necrosis but is more commonly associated with apoptosis. During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell. XIAP is the most potent caspase inhibitor encoded in the mammalian genome. The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells. CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation. In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin. Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs. Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment. Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases. Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments. With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns. While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus. In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol. Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines.

KDEL-Cargo Regulates Interactions between Proteins Involved in COPI Vesicle Traffic: Measurements in Living Cells Using FRET

How the occupied KDEL receptor ERD2 is sorted into COPI vesicles for Golgi-to-ER transport is largely unknown. Here, interactions between proteins of the COPI transport machinery occurring during a “wave” of transport of a KDEL ligand were studied in living cells. FRET between CFP and YFP fusion proteins was measured by multifocal multiphoton microscopy and bulk-cell spectrofluorimetry. Ligand binding induces oligomerization of ERD2 and recruitment of ARFGAP to the Golgi, where the (ERD2) n/ARFGAP complex interacts with membrane-bound ARF1. During KDEL ligand transport, interactions of ERD2 with β-COP and p23 decrease and the proteins segregate. Both p24a and p23 interact with ARF1, but only p24 interacts with ARFGAP. These findings suggest a model for how cargo-induced oligomerization of ERD2 regulates its sorting into COPI-coated buds.

Dimerization of receptor protein-tyrosine phosphatase alpha in living cells.

BackgroundDimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.ResultsIn order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.ConclusionsWe demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

Dynamic Analyses of the Expression of the HISTONE::YFP Fusion Protein in Arabidopsis Show That Syncytial Endosperm Is Divided in Mitotic Domains

Dynamic Analyses of the Expression of the HISTONE::YFP Fusion Protein in Arabidopsis Show That Syncytial Endosperm Is Divided in Mitotic Domains