Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum.

When the K+ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.

Toward Fabrication of Bioactive Papers: Covalent Immobilization of Peptides and Proteins.

Herein, we report a novel two-step method for the covalent, site-directed, and efficient immobilization of proteins on lab-made paper sheets. First, paper fibers were modified with a peptidic anchor comprising enzyme recognition motifs. Four different conjugation strategies for peptide immobilization were evaluated with respect to reproducibility and fiber loading efficiency. After manufacturing of the peptide-preconditioned paper, oriented conjugation of the model protein tGFP containing a C-terminal recognition sequence for either sortase A or microbial transglutaminase was assessed semiquantitatively by fluorescence measurement and inspected by confocal laser scanning microscopy (CLSM). The two enzymes utilized for protein conjugation used the same oligoglycine peptide anchor, and both proved to be suitable for controlled oriented linkage of substrate proteins at physiological conditions.

Reducing Unspecific Protein Adsorption in Microfluidic Papers Using Fiber-Attached Polymer Hydrogels.

Microfluidic paper combines pump-free water transport at low cost with a high degree of sustainability, as well as good availability of the paper-forming cellulosic material, thus making it an attractive candidate for point-of-care (POC) analytics and diagnostics. Although a number of interesting demonstrators for such paper devices have been reported to date, a number of challenges still exist, which limit a successful transfer into marketable applications. A strong limitation in this respect is the (unspecific) adsorption of protein analytes to the paper fibers during the lateral flow assay. This interaction may significantly reduce the amount of analyte that reaches the detection zone of the microfluidic paper-based analytical device (µPAD), thereby reducing its overall sensitivity. Here, we introduce a novel approach on reducing the nonspecific adsorption of proteins to lab-made paper sheets for the use in µPADs. To this, cotton linter fibers in lab-formed additive-free paper sheets are modified with a surrounding thin hydrogel layer generated from photo-crosslinked, benzophenone functionalized copolymers based on poly-(oligo-ethylene glycol methacrylate) (POEGMA) and poly-dimethyl acrylamide (PDMAA). This, as we show in tests similar to lateral flow assays, significantly reduces unspecific binding of model proteins. Furthermore, by evaporating the transport fluid during the microfluidic run at the end of the paper strip through local heating, model proteins can almost quantitatively be accumulated in that zone. The possibility of complete, almost quantitative protein transport in a µPAD opens up new opportunities to significantly improve the signal-to-noise (S/N) ratio of paper-based lateral flow assays.

An Exo2 derivative affects ER and Golgi morphology and vacuolar sorting in a tissue-specific manner in arabidopsis.

We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 µm, the effects of LG8 are reversible.

PDMP induces rapid changes in vacuole morphology in Arabidopsis root cells.

PDMP (D-L-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol) is a well-known inhibitor of glucosylceramide synthase (GCS), a key enzyme in sphingolipid biosynthesis. Through the resultant increase in ceramides which interact with mTOR and Beclin1 (Atg6), this drug is also known to induce macroautophagy in mammalian cells. This study investigated the response of Arabidopsis root cells to PDMP, and what are probably numerous tightly packed small vacuoles in the control cells appear to fuse to form a single globular-shaped vacuole. However, during this fusion process, cytoplasm channels between the individual vacuoles become trapped in deep invaginations of the tonoplast. In both optical sections in the confocal laser scanning microscope and in ultrathin sections in the electron microscope, these invaginations have the appearance of cytoplasmic inclusions in the vacuole lumen. These changes in vacuole morphology are rapid (occurring within minutes after application of PDMP) and are independent of ongoing protein synthesis. The tonoplast invaginations remain visible for hours, but after 24h almost all disappear. Experiments designed to examine whether ceramide levels might be the cause of the PDMP effect have not proved conclusive. On the other hand, this study has been able to rule out the release of Ca(2+) ions from intracellular stores as a contributing factor.

Putative p24 complexes in Arabidopsis contain members of the delta and beta subfamilies and cycle in the early secretory pathway.

p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24ß, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24ß and p24δ subfamilies, the latter probably including two different subclasses. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 (p24δ1 subclass) localizes to the endoplasmic reticulum (ER) at steady state as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. It is now shown that transiently expressed RFP-p24δ9 (p24δ2 subclass) also localizes to the ER. In contrast, transiently expressed green fluorescent protein (GFP)-p24ß3 mainly localizes to the Golgi apparatus (as p24ß2) and exits the ER in a COPII-dependent manner. Immunogold electron microscopy in Arabidopsis root tip cells using specific antibodies shows that endogenous p24δ9 localizes mainly to the ER but also partially to the cis-Golgi. In contrast, endogenous p24ß3 mainly localizes to the Golgi apparatus. By a combination of experiments using transient expression, knock-out mutants, and co-immunoprecipitation, it is proposed that Arabidopsis p24 proteins form different heteromeric complexes (including members of the ß and δ subfamilies) which are important for their stability and their coupled trafficking at the ER-Golgi interface. Evidence is also provided for a role for p24δ5 in retrograde Golgi-ER transport of the KDEL-receptor ERD2.

Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle.

Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

Comparison of membrane targeting strategies for the accumulation of the human immunodeficiency virus p24 protein in transgenic tobacco.

Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.

Coupled transport of Arabidopsis p24 proteins at the ER-Golgi interface.

p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24ß, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24ß and p24δ subfamilies. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24δ5 localizes to the endoplasmic reticulum (ER) as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. Using specific antibodies, endogenous p24δ5 has now been localized to the ER and p24ß2 to the Golgi apparatus in Arabidopsis root tip cells by immunogold electron microscopy. The relative contributions of the cytosolic tail and the luminal domains to p24δ5 trafficking have also been characterized. It is demonstrated that whereas the dilysine motif in the cytoplasmic tail determines the location of p24δ5 in the early secretory pathway, the luminal domain may contribute to its distribution downstream of the Golgi apparatus. By using knock-out mutants and co-immunoprecipitation experiments, it is shown that p24δ5 and p24ß2 interact with each other. Finally, it is shown that p24δ5 and p24ß2 exhibit coupled trafficking at the ER-Golgi interface. It is proposed that p24δ5 and p24ß2 interact with each other at ER export sites for ER exit and coupled transport to the Golgi apparatus. Once in the Golgi, p24δ5 interacts very efficiently with the COPI machinery for retrograde transport back to the ER.

Modulation of Differentiation and Bone Resorbing Activity of Human (Pre-) Osteoclasts After X-Ray Exposure.

Low-dose radiotherapy (LD-RT) is a local treatment option for patients with chronic degenerative and inflammatory diseases, in particular musculoskeletal diseases. Despite reported analgesic and anti-inflammatory effects, cellular and molecular mechanisms related to osteoimmunological effects are still elusive. Here we test the hypothesis that X-irradiation inhibits the differentiation of precursor osteoclasts into mature osteoclasts (mOC) and their bone resorbing activity. Circulating monocytes from healthy donors were isolated and irradiated after attachment with single or fractionated X-ray doses, comparable to an LD-RT treatment scheme. Then monocytes underwent ex vivo differentiation into OC during cultivation up to 21 days, under conditions mimicking the physiological microenvironment of OC on bone. After irradiation, apoptotic frequencies were low, but the total number of OC precursors and mOC decreased up to the end of the cultivation period. On top, we observed an impairment of terminal differentiation, i.e. a smaller fraction of mOC, reduced resorbing activity on bone, and release of collagen fragments. We further analyzed the effect of X-irradiation on multinucleation, resulting from the fusion of precursor OC, which occurs late during OC differentiation. At 21 days after exposure, the observation of smaller cellular areas and a reduced number of nuclei per mOC suggest an impaired fusion of OC precursors to form mOC. Before, at 14 days, the nuclear translocation of Nuclear Factor Of Activated T Cells 1 (NFATc1), a master regulator of osteoclast differentiation and fusion, was decreased. In first results, obtained in the frame of a longitudinal LD-RT study, we previously reported a pain-relieving effect in patients. However, in a subgroup of patients suffering from Calcaneodynia or Achillodynia, we did not observe a consistent decrease of established blood markers for resorption and formation of bone, or modified T cell subtypes involved in regulating these processes. To assess the relevance of changes in bone metabolism for other diseases treated with LD-RT will be subject of further studies. Taken together, we observed that in vitro X-irradiation of monocytes results in an inhibition of the differentiation into bone-resorbing OC and a concomitant reduction of resorbing activity. The detected reduced NFATc1 signaling could be one underlying mechanism.

Retromer recycles vacuolar sorting receptors from the trans-Golgi network.

Receptor-mediated sorting processes in the secretory pathway of eukaryotic cells rely on mechanisms to recycle the receptors after completion of transport. Based on this principle, plant vacuolar sorting receptors (VSRs) are thought to recycle after dissociating of receptor-ligand complexes in a pre-vacuolar compartment. This recycling is mediated by retromer, a cytosolic coat complex that comprises sorting nexins and a large heterotrimeric subunit. To analyse retromer-mediated VSR recycling, we have used a combination of immunoelectron and fluorescence microscopy to localize the retromer components sorting nexin 1 (SNX1) and sorting nexin 2a (SNX2a) and the vacuolar sorting protein VPS29p. All retromer components localize to the trans-Golgi network (TGN), which is considered to represent the early endosome of plants. In addition, we show that inhibition of retromer function in vivo by expression of SNX1 or SNX2a mutants as well as transient RNAi knockdown of all sorting nexins led to accumulation of the VSR BP80 at the TGN. Quantitative protein transport studies and live-cell imaging using fluorescent vacuolar cargo molecules revealed that arrival of these VSR ligands at the vacuole is not affected under these conditions. Based on these findings, we propose that the TGN is the location of retromer-mediated recycling of VSRs, and that transport towards the lytic vacuole downstream of the TGN is receptor-independent and occurs via maturation, similar to transition of the early endosome into the late endosome in mammalian cells.

Sorting of plant vacuolar proteins is initiated in the ER.

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor-ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer-mediated recycling of the plant VSR BP80 starts at the trans-Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co-expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII-mediated transport route. Retention of soluble cargo despite ongoing COPII-mediated bulk flow can only be explained by an interaction with membrane-bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER-anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR-ligand interaction. It also implies that the retromer-mediated recycling route for the VSRs leads from the TGN back to the ER.

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported.

Differential effects of the brefeldin A analogue (6R)-hydroxy-BFA in tobacco and Arabidopsis.

The effects of two brefeldin A (BFA) analogues (BFA lactam; 6(R)-hydroxy-BFA) on plant cells were tested. Although these two compounds elicited BFA-like effects in mammalian cells, the lactam analogue failed to elicit a response in plant cells. By contrast, while the 6(R)-hydroxy-BFA analogue gave rise to a classic BFA response in tobacco mesophyll protoplasts and true leaves of Arabidopsis (redistribution of Golgi enzymes into the ER), it failed to cause the formation of BFA-compartments in Arabidopsis root cells and cotyledonary leaves. Even when the GNL1-LM mutant of Arabidopsis, which has a cis-Golgi located BFA-sensitive ARF-GEF, was used, the 6(R)-hydroxy analogue failed to elicit a response at conventional BFA concentrations. Only at concentrations of over 200 µM did 6(R)-hydroxy-BFA elicit a BFA-like effect. These differences are interpreted in terms of the different properties of the respective TGN- (Arabidopsis roots) and cis-Golgi- (tobacco mesophyll) localized BFA-sensitive ARF-GEFs.

Codon Bias Can Determine Sorting of a Potassium Channel Protein.

Due to the redundancy of the genetic code most amino acids are encoded by multiple synonymous codons. It has been proposed that a biased frequency of synonymous codons can affect the function of proteins by modulating distinct steps in transcription, translation and folding. Here, we use two similar prototype K+ channels as model systems to examine whether codon choice has an impact on protein sorting. By monitoring transient expression of GFP-tagged channels in mammalian cells, we find that one of the two channels is sorted in a codon and cell cycle-dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves, together with a cell-state-dependent decoding mechanism, as a secondary code for sorting intracellular membrane proteins.

ROS- and Radiation Source-Dependent Modulation of Leukocyte Adhesion to Primary Microvascular Endothelial Cells.

Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose-effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte adhesion. Here, we evaluated the expression of anti-oxidative enzymes and the transcription factor Nrf2 (Nuclear factor-erythroid-2-related factor 2), intracellular ROS content, and leukocyte adhesion in primary human microvascular endothelial cells (HMVEC) upon low-dose irradiation under physiological laminar shear stress or static conditions after irradiation with X-ray or Carbon (C)-ions (0-2 Gy). Laminar conditions contributed to increased mRNA expression of anti-oxidative factors and reduced ROS in HMVEC following a 0.1 Gy X-ray and 0.5 Gy C-ion exposure, corresponding to reduced leukocyte adhesion and expression of adhesion molecules. By contrast, mRNA expression of anti-oxidative markers and adhesion molecules, ROS, and leukocyte adhesion were not altered by irradiation under static conditions. In conclusion, irradiation of endothelial cells with low doses under physiological laminar conditions modulates the mRNA expression of key factors of the anti-oxidative system, the intracellular ROS contents of which contribute at least in part to leucocyte adhesion, dependent on the radiation source.

Carbohydrate binding module-fused antibodies improve the performance of cellulose-based lateral flow immunoassays.

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.

In vivo trafficking and localization of p24 proteins in plant cells.

p24 proteins constitute a family of putative cargo receptors that traffic in the early secretory pathway. p24 proteins can be divided into four subfamilies (p23, p24, p25 and p26) by sequence homology. In contrast to mammals and yeast, most plant p24 proteins contain in their cytosolic C-terminus both a dilysine motif in the -3, -4 position and a diaromatic motif in the -7, -8 position. We have previously shown that the cytosolic tail of Arabidopsis p24 proteins has the ability to interact with ARF1 and coatomer (through the dilysine motif) and with COPII subunits (through the diaromatic motif). Here, we establish the localization and trafficking properties of an Arabidopsis thaliana p24 protein (Atp24) and have investigated the contribution of the sorting motifs in its cytosolic tail to its in vivo localization. Atp24-red fluorescent protein localizes exclusively to the endoplasmic reticulum (ER), in contrast with the localization of p24 proteins in other eukaryotes, and the dilysine motif is necessary and sufficient for ER localization. In contrast, Atp24 mutants lacking the dilysine motif are transported along the secretory pathway to the prevacuolar compartment and the vacuole, although a significant fraction is also found at the plasma membrane. Finally, we have found that ER export of Atp24 is COPII dependent, while its ER localization requires COPI function, presumably for efficient Golgi to ER recycling.

The syntaxins SYP31 and SYP81 control ER-Golgi trafficking in the plant secretory pathway.

Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter alpha-amylase with the ER-retained reporter alpha-amylase-HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N-terminal-tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C-terminal-tagged SYP31 failed to exhibit this effect. Overexpression of both wild-type and N-terminal-tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)-SYP31 was still visible as fluorescent punctae, which, unlike SYP31-GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non-inhibitory levels, YFP-SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites.

BFA effects are tissue and not just plant specific.

Brefeldin A (BFA) is one of the most popular drugs used by researchers for studies on secretion and endocytosis because it interferes with specific vesicle coat proteins via action on a guanine nucleotide exchange factor. Due to its range of morphological effects on the Golgi apparatus in a variety of plant tissues, we believe that there is more to the BFA response than the primary molecular targets so far identified.