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Petra Boevink

Publications and source records attributed to Petra Boevink.

6 recordsLinked to original sources
PubMed2006

Targeting of TMV movement protein to plasmodesmata requires the actin/ER network: evidence from FRAP.

Fluorescence recovery after photobleaching (FRAP) was used to study the mechanism by which fluorescent-protein-tagged movement protein (MP) of tobacco mosaic virus (TMV) is targeted to plasmodesmata (PD). The data show that fluorescence recovery in PD at the leading edge of an infection requires elements of the cortical actin/endoplasmic reticulum (ER) network and can occur in the absence of an intact microtubule (MT) cytoskeleton. Inhibitors of the actin cytoskeleton (latrunculin and cytochalasin) significantly inhibited MP targeting, while MT inhibitors (colchicine and oryzalin) did not. Application of sodium azide to infected cells implicated an active component of MP transfer to PD. Treatment of cells with Brefeldin A (BFA) at a concentration that caused reabsorption of the Golgi bodies into the ER (precluding secretion of viral MP) had no effect on MP targeting, while disruption of the cortical ER with higher concentrations of BFA caused significant inhibition. Our results support a model of TMV MP function in which targeting of MP to PD during infection is mediated by the actin/ER network.

Actins↗
PubMed2005

An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1.

GRIP domain proteins are a class of golgins that have been described in yeast and animals. They locate to the trans-Golgi network and are thought to play a role in endosome-to-Golgi trafficking. The Arabidopsis GRIP domain protein, AtGRIP, fused to the green fluorescent protein (GFP), locates to Golgi stacks but does not exactly co-locate with the Golgi marker sialyl transferase (ST)-mRFP, nor with the t-SNAREs Memb11, SYP31 and BS14a. We conclude that the location of AtGRIP is further to the trans side of the stack than STtmd-mRFP. The 185-aa C-terminus of AtGRIP containing the GRIP domain targeted GFP to the Golgi, although a proportion of the fusion protein was still found in the cytosol. Mutation of a conserved tyrosine (Y717) to alanine in the GRIP domain disrupted Golgi localization. ARL1 is a small GTPase required for Golgi targeting of GRIP domain proteins in other systems. An Arabidopsis ARL1 homologue was isolated and shown to target to Golgi stacks. The GDP-restricted mutant of ARL1, AtARL1-T31N, was observed to locate partially to the cytosol, whereas the GTP-restricted mutant AtARL1-Q71L labelled the Golgi and a population of small structures. Increasing the levels of AtARL1 in epidermal cells increased the proportion of GRIP-GFP fusion protein on Golgi stacks. We show, moreover, that AtARL1 interacted with the GRIP domain in a GTP-dependent manner in vitro in affinity chromatography and in the yeast two-hybrid system. This indicates that AtGRIP and AtARL1 interact directly. We conclude that the pathway involving ARL1 and GRIP domain golgins is conserved in plants.

Amino Acid Sequence↗
PubMed2003

ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants.

Quality control in the secretory pathway is a fundamental step in preventing deleterious effects that may arise by the release of malfolded proteins into the cell or apoplast. Our aims were to visualise and analyse the disposal route followed by aberrant proteins within a plant cell in vivo using fluorescent protein technology. A green fluorescent protein (GFP) fusion was detected in the cytosol and the nucleoplasm in spite of the presence of an N-terminal secretory signal peptide. In contrast to secreted GFP, the fusion protein was retained in the cells where it was degraded slowly, albeit at a rate much higher than that of the endoplasmic reticulum (ER)-retained derivative GFP-HDEL. The fusion protein could not be stabilised by inhibitors of transport or the cytosolic proteasome. However, the protein is a strong lumenal binding protein (BiP) ligand. Complete signal peptide processing even after long-term expression in virus-infected leaves rules out the possibility that the documented accumulation in the cytosol and nucleoplasm is because of the bypassing of the translocation pores. The data are consistent with the hypothesis that the fusion protein is disposed off from the ER via a retrograde translocation back to the cytosol. Moreover, accumulation in the nucleoplasm was shown to be microtubule dependent unlike the well-documented diffusion of cytosolically expressed GFP into the nucleoplasm. The apparent active transport of the GFP fusion into the nucleoplasm may indicate an as yet undiscovered feature of the ER-associated degradation (ERAD) pathway and explain the insensitivity to degradation by proteasome inhibitors.

Biological Transport↗
PubMed2003

High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata.

A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana. Libraries of random, partial cDNAs fused to the 5' or 3' end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus. Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5' end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of >20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles.

Base Sequence↗
PubMed2002

Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus.

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.

DNA, Viral↗