Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca 2؉ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca 2؉ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca 2؉ into the stores. Measurement of intra-organelle-free Ca 2؉ in permeabilized cells revealed that the ability of 2B to reduce the Ca 2؉ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cellfree system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca 2؉ -ATPase that transports Ca 2؉ from the cytosol into the stores. To investigate whether 2B specifically affects Ca 2؉ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H ؉ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.Enteroviruses (e.g. poliovirus, coxsackievirus, ECHOvirus) belong to the family of Picornaviridea, a large family of nonenveloped, cytolytic viruses that contain a single-stranded RNA genome of positive polarity. Upon infection, enteroviruses induce a number of dramatic alterations in their host cell, which serve to create the appropriate conditions for viral RNA replication and/or prevent antiviral host cell responses. One of these alterations is the modification of intracellular Ca 2ϩ homeostasis. We have previously shown that infection of HeLa cells with coxsackievirus results in a reduction of the amount of Ca 2ϩ that can be released from the intracellular stores using thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), 2 the Ca 2ϩ -ATPase that transports Ca 2ϩ from the cytosol into the stores. In addition, a gradual increase in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) was observed due to the influx of extracellular Ca 2ϩ (1). The enterovirus 2B protein, one of the nonstructural proteins involved in viral RNA replication, plays a major role in the alterations in intracellular Ca 2ϩ homeostasis that take place in enterovirus-infected cells (1, 2). The mechanism by which 2B, or its precursor 2BC, exerts its effects is largely unknown. Ca 2ϩ homeostasis in the intracellular stores (i.e. endoplasmic reticulum (ER) and Golgi) is the net result of the activity of the SERCA on the one hand and the continuous passive Ca 2ϩ leak from these organelles that exists under normal conditions on the other hand (3). Thus, the reductions in the Ca 2ϩ filling state of the stores ...

Mitochondrial isolated complex I deficiency is the most frequently encountered OXPHOS defect. We report a patient with an isolated complex I deficiency expressed in skin fibroblasts as well as muscle tissue. Because the parents were consanguineous, we performed homozygosity mapping to identify homozygous regions containing candidate genes such as NDUFA2 on chromosome 5. Screening of this gene on genomic DNA revealed a mutation that interferes with correct splicing and results in the skipping of exon 2. Exon skipping was confirmed on the mRNA level. The mutation in this accessory subunit causes reduced activity and disturbed assembly of complex I. Furthermore, the mutation is associated with a mitochondrial depolarization. The expression and activity of complex I and the depolarization was (partially) rescued with a baculovirus system expressing the NDUFA2 gene.

The role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H ؉ ,K ؉ -ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the ␣-

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,KATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (

Intracellular chemical reactions generally constitute reaction-diffusion systems located inside nanostructured compartments like the cytosol, nucleus, endoplasmic reticulum, Golgi, and mitochondrion. Understanding the properties of such systems requires quantitative information about solute diffusion. Here we present a novel approach that allows determination of the solvent-dependent solute diffusion constant (D solvent ) inside cell compartments with an experimentally quantifiable nanostructure. In essence, our method consists of the matching of synthetic fluorescence recovery after photobleaching (FRAP) curves, generated by a mathematical model with a realistic nanostructure, and experimental FRAP data. As a proof of principle, we assessed D solvent of a monomeric fluorescent protein (AcGFP1) and its tandem fusion (AcGFP1 2 ) in the mitochondrial matrix of HEK293 cells. Our results demonstrate that diffusion of both proteins is substantially slowed by barriers in the mitochondrial matrix (cristae), suggesting that cells can control the dynamics of biochemical reactions in this compartment by modifying its nanostructure. molecular dynamics | quantitative random-walk model | systems biology A major challenge facing biochemistry is to understand the dynamics of chemical reactions within inhomogeneous cell compartments like the cytosol, nucleus, endoplasmic reticulum (ER), Golgi, and mitochondrion (1). In general, intracompartment reactions involve the conversion of (im)mobile substrates by (im)mobile enzymes into (im)mobile products and therefore constitute reaction-diffusion systems. Obviously, gaining insight into the behavior of such systems requires quantitative information about solute diffusion. The latter depends on solvent and solute properties, the dimensions and shape of the compartment, and the internal structure of the compartment (2-6).A widely used strategy to investigate solute diffusion involves expressing a fluorescent tracer protein (FP) in the compartment of interest. Next, FP mobility is measured using FCS (fluorescence correlation spectroscopy) or FRAP (fluorescence recovery after photobleaching). This is then followed by curve fitting and/ or mathematical modeling of the experimental data to obtain the diffusion constant of the FP (7-16). However, these analysis methods generally do not include realistic (i.e., experimentally determined) information concerning the spatial dimensions and nanostructure of the compartment. Moreover, the temporal scale of most FRAP models does not quantitatively match with that of FRAP experiments. Therefore it was already recognized some time ago (8-17) that the above approaches will only yield an "apparent" (biased) value for the diffusion constant (D app ) of a given FP, which represents an underestimation of the "real" (i.e., purely solvent-dependent) diffusion constant (D solvent ).In this study we present a strategy to determine D solvent inside cell compartments with an experimentally accessible nanostructure. Our method consists of matching synthetic FRA...