Assessment of the accuracy of estimated free phenytoin concentrations in a mixed patient population.

Objectives: Phenytoin is an anti-epileptic drug that has a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Only the free fraction is pharmacologically active, and in some cases, accurate determination of the free phenytoin concentration may be essential to prevent phenytoin toxicity. Although it is possible to measure free phenytoin concentration, often only the total concentration is measured, with equations used to estimate the free fraction.

An enzymatic assay for the detection of glycolic acid in serum as a marker of ethylene glycol poisoning.

Background: Ingestion of ethylene glycol is a relatively rare event but one with potentially lethal consequences. Early diagnosis and appropriate treatment are essential. However, diagnosis of poisoning can only be confirmed definitively by the measurement of ethylene glycol and/or its metabolites, usually performed by gas chromatographic methods.

Efficient mitochondrial targeting relies on co-operation of multiple protein signals in plants.

To date, the most prevalent model for transport of pre-proteins to plant mitochondria is based on the activity of an N-terminal extension serving as a targeting peptide. Whether the efficient delivery of proteins to mitochondria is based exclusively on the action of the N-terminal extension or also on that of other protein determinants has yet to be defined. A novel mechanism is reported here for the targeting of a plant protein, named MITS1, to mitochondria.

Dynamic organization of COPII coat proteins at endoplasmic reticulum export sites in plant cells.

Protein export from the endoplasmic reticulum (ER) is mediated by the accumulation of COPII proteins such as Sar1, Sec23/24 and Sec13/31 at specialized ER export sites (ERES). Although the distribution of COPII components in mammalian and yeast systems is established, a unified model of ERES dynamics has yet to be presented in plants. To investigate this, we have followed the dynamics of fluorescent fusions to inner and outer components of the coat, AtSec24 and AtSec13, in three different plant model systems: tobacco and Arabidopsis leaf epidermis, as well as tobacco BY-2 suspension cells.

Plant Sar1 isoforms with near-identical protein sequences exhibit different localisations and effects on secretion.

In plants, differentiation of subdomains of the endoplasmic reticulum (ER) dedicated to protein export, the ER export sites (ERES), is influenced by the type of export-competent membrane cargo to be delivered to the Golgi. This raises a fundamental biological question: is the formation of transport intermediates at the ER for trafficking to the Golgi always regulated in the same manner? To test this, we followed the distribution and activity of two plant Sar1 isoforms. Sar1 is the small GTPase that regulates assembly of COPII (coat protein complex II) on carriers that transport secretory cargo from ER to Golgi.

Correct targeting of plant ARF GTPases relies on distinct protein domains.

Indispensable membrane trafficking events depend on the activity of conserved small guanosine triphosphatases (GTPases), anchored to individual organelle membranes. In plant cells, it is currently unknown how these proteins reach their correct target membranes and interact with their effectors. To address these important biological questions, we studied two members of the ADP ribosylation factor (ARF) GTPase family, ARF1 and ARFB, which are membrane anchored through the same N-terminal myristoyl group but to different target membranes.

Studying protein export from the endoplasmic reticulum in plants.

Understanding the mechanisms of protein sorting and targeting through the plant secretory pathway has become the focus of many research laboratories. The development of a model system whereby recombinant genes can be transiently expressed in protoplasts has facilitated the study of protein transport signals. Experimental strategies combining a protoplast expression system with endoglycosidase H, vacuole purification, and pulse-chase analyses are used to investigate aspects of specific proteins as they pass through the secretory system.

Localization and topogenesis studies of cytoplasmic and vacuolar homologs of the Galanthus nivalis agglutinin.

The Galanthus nivalis agglutinin (GNA) is synthesized as a preproprotein. To corroborate the role of the different targeting peptides in the topogenesis of GNA and related proteins, different constructs were made whereby both the complete original GNA gene and different truncated sequences were coupled to the enhanced green fluorescent protein (EGFP). In addition, a GNA ortholog from rice that lacks the signal peptide and C-terminal propeptide sequence was fused to EGFP.

Post-Golgi protein traffic in the plant secretory pathway.

The Golgi apparatus in plants is organized as a multitude of individual stacks that are motile in the cytoplasm and in close association with the endoplasmic reticulum (ER) (Boevink et al. in Plant J 15:441-447, 1998). These stacks operate as a sorting centre for cargo molecules, providing modification and redirection to other organelles as appropriate.

De novo formation of plant endoplasmic reticulum export sites is membrane cargo induced and signal mediated.

The plant endoplasmic reticulum (ER) contains functionally distinct subdomains at which cargo molecules are packed into transport carriers. To study these ER export sites (ERES), we used tobacco (Nicotiana tabacum) leaf epidermis as a model system and tested whether increased cargo dosage leads to their de novo formation. We have followed the subcellular distribution of the known ERES marker based on a yellow fluorescent protein (YFP) fusion of the Sec24 COPII coat component (YFP-Sec24), which, differently from the previously described ERES marker, tobacco Sar1-YFP, is visibly recruited at ERES in both the presence and absence of overexpressed membrane cargo.

Multiple roles of ADP-ribosylation factor 1 in plant cells include spatially regulated recruitment of coatomer and elements of the Golgi matrix.

Recent evidence indicates that ADP-ribosylation factor 1 (ARF1) carries out multiple roles in plant cells that may be independent from the established effector complex COPI. To investigate potential COPI-independent functions, we have followed the dynamics of ARF1 and a novel putative effector, the plant golgin GRIP-related ARF-binding domain-containing Arabidopsis (Arabidopsis thaliana) protein 1 (GDAP1) in living plant cells. We present data that ascribe a new role to ARF1 in plant cell membrane traffic by showing that the GTPase functions to recruit GDAP1 to membranes.

Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond.

Significant advances have been made in recent years that have increased our understanding of the trafficking to and from membranes that are functionally linked to the Golgi apparatus in plants. New routes from the Golgi to organelles outside the secretory pathway are now being identified, revealing the importance of the Golgi apparatus as a major sorting station in the plant cell. This review discusses our current perception of Golgi structure and organization as well as the molecular mechanisms that direct traffic in and out of the Golgi.

ARL1 plays a role in the binding of the GRIP domain of a peripheral matrix protein to the Golgi apparatus in plant cells.

ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1.

Seeking a way out: export of proteins from the plant endoplasmic reticulum.

The functionality of the secretory pathway relies on the efficient transfer of cargo molecules from their site of synthesis in the endoplasmic reticulum (ER) to successive compartments within the pathway. Although transport mechanisms of secretory proteins have been studied in detail in various non-plant systems, it is only recently that our knowledge of secretory routes in plants has expanded dramatically. This review focuses on exciting new findings concerning the exit mechanisms of cargo proteins from the plant ER and the role of ER export sites in this process.

Mapping the Arabidopsis organelle proteome.

A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments.

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery.

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized.

Fluorescent proteins as markers in the plant secretory pathway.

The use of fluorescent proteins and live cell imaging has greatly increased our knowledge of cell biology in recent years. Not only can these technologies be used to study protein trafficking under different conditions, but they have also been of use in elucidating the relationships between different organelles in a noninvasive manner. The use of multiple different fluorochromes allows the observation of interactions between organelles and between proteins, making this one of the fastest-developing and exciting fields at this time.

Diacidic motifs influence the export of transmembrane proteins from the endoplasmic reticulum in plant cells.

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins.

Identification and characterization of AtCASP, a plant transmembrane Golgi matrix protein.

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin.

Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants.

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented.

Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting.

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting.

The GTPase ARF1p controls the sequence-specific vacuolar sorting route to the lytic vacuole.

We have studied the transport of soluble cargo molecules by inhibiting specific transport steps to and from the Golgi apparatus. Inhibition of export from the Golgi via coexpression of a dominant-negative GTP-restricted ARF1 mutant (Q71L) inhibits the secretion of alpha-amylase and simultaneously induces the secretion of the vacuolar protein phytepsin to the culture medium. By contrast, specific inhibition of endoplasmic reticulum export via overexpression of Sec12p or coexpression of a GTP-restricted form of Sar1p inhibits the anterograde transport of either cargo molecule in a similar manner.