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1.  Application of the gene dosage balance hypothesis to auxin-related ribosomal mutants in Arabidopsis 
Plant Signaling & Behavior  2010;5(4):450-452.
Several proteomic studies in Arabidopsis have shown the presence of heterogeneous ribosomal populations in different tissues. However, the phenotypic consequences of the imbalance of those ribosomal populations, and the regulatory mechanisms activated to control specific ratios between them, have yet to be evaluated. In our previous report, the phenotypic characterization of the ribosomal protein family L4 (RPL4) in Arabidopsis suggests that the maintenance of proper auxin-regulated developmental responses requires the simultaneous presence of RPL4A- and RPL4D-containing ribosomes. Based on the analysis of the compensatory mechanisms within the RPL4 family proteins in the rpl4a and rpl4d backgrounds, we propose the Gene Dosage Balance Hypothesis (GDBH) as a regulatory mechanism for ribosomal complexes in Arabidopsis. By using the concepts of dosage compensation and hierarchy, GDBH is able to explain the severity and specificity of different ribosomal mutant phenotypes associated with the same ribosomal complex.
PMCID: PMC2958597  PMID: 20383066
ribosomal complex; gene dosage balance hypothesis; auxins; Arabidopsis thaliana; ribosomal protein family RPL4
2.  Chemical dissection of endosomal pathways 
Plant Signaling & Behavior  2009;4(1):57-62.
Membrane trafficking and associated signal transduction pathways are critical for plant development and responses to environment. These transduction pathways, including those for brassinosteroids and auxins, require endocytosis to endosomes and recycling back to the plasma membrane. A major challenge toward understanding these processes and their biological roles has been the highly dynamic nature of endomembrane trafficking. To effectively study endocytosis and recycling, which occur in a time frame of minutes, bioactive chemicals provide a powerful and exacting tool. Pharmacological inhibitors such as Brefeldin A (BFA) and the newly identified Endosidin 1 (ES1) have been used to define endosome compartments. ES1 is a clear example of the ability of chemicals to dissect even distinct subpopulations of endosomes involved in trafficking and signal transduction. The ability to characterize and dissect such highly dynamic pathways in a temporal and spatial manner is possible only using pharmacological reagents which can act rapidly and reversibly.
PMCID: PMC2634075  PMID: 19704710
endocytosis; endosome; endosidin 1; brassinosteroid; brefeldin A; ARF-GEF; SYP61; VHA-a1
3.  Powerful Partners: Arabidopsis and Chemical Genomics 
Chemical genomics (i.e. genomics scale chemical genetics) approaches capitalize on the ability of low molecular mass molecules to modify biological processes. Such molecules are used to modify the activity of a protein or a pathway in a manner that it is tunable and reversible. Bioactive chemicals resulting from forward or reverse chemical screens can be useful in understanding and dissecting complex biological processes due to the essentially limitless variation in structure and activities inherent in chemical space. A major advantage of this approach as a powerful addition to conventional plant genetics is the fact that chemical genomics can address loss-of-function lethality and redundancy. Furthermore, the ability of chemicals to be added at will and to act quickly can permit the study of processes that are highly dynamic such as endomembrane trafficking. An important aspect of utilizing small molecules effectively is to characterize bioactive chemicals in detail including an understanding of structure-activity relationships and the identification of active and inactive analogs. Bioactive chemicals can be useful as reagents to probe biological pathways directly. However, the identification of cognate targets and their pathways is also informative and can be achieved by screens for genetic resistance or hypersensitivity in Arabidopsis thaliana or other organisms from which the results can be translated to plants. In addition, there are approaches utilizing “tagged” chemical libraries that possess reactive moieties permitting the immobilization of active compounds. This opens the possibility for biochemical purification of putative cognate targets. We will review approaches to screen for bioactive chemicals that affect biological processes in Arabidopsis and provide several examples of the power and challenges inherent in this new approach in plant biology.
doi:10.1199/tab.0109
PMCID: PMC3243329  PMID: 22303245
4.  Identification of cellular pathways affected by Sortin2, a synthetic compound that affects protein targeting to the vacuole in Saccharomyces cerevisiae 
Background
Sortin2 is a low mass compound that interferes with vacuolar delivery of proteins in plants and yeast. The Sortin2 phenotype was tested in Arabidopsis thaliana and found to be reversible upon drug removal, demonstrating the ability of chemical genomics to induce reversible phenotypes that would be difficult to achieve using conventional genetics [1]. However, standard genetic methods can be used to identify drug target pathways in a high-throughput manner.
Results
In this study, we analyzed structure-function relationships of Sortin2 using structural analogues. The results show the key roles of sulphite substitution and a benzoic acid group. A Sortin 2 hypersensitivity screen for the induced secretion of a vacuolar cargo protein was done utilizing a yeast haploid deletion library. Using bioinformatics approaches, we highlighted functional information about the cellular pathways affected by drug treatment which included protein sorting and other endomembrane system-related processes.
Conclusion
Chemical, genomic and genetics approaches were used to understand the mode of action of Sortin2, a bioactive chemical that affects the delivery of a vacuolar protein. Critical features of Sortin2 structure necessary for bioactivity suggest a binding pocket that may recognize two ends of Sortin2. The genome-wide screen shows that Sortin2 treatment in yeast affects primarily components within the endomembrane system. This approach allowed us to assign putative functions in protein sorting for fifteen genes of previously unknown function.
doi:10.1186/1472-6769-8-1
PMCID: PMC2265672  PMID: 18179719
5.  The AtC–VPS Protein Complex Is Localized to the Tonoplast and the Prevacuolar Compartment in Arabidopsis 
Molecular Biology of the Cell  2003;14(2):361-369.
Plant cells contain several types of vacuoles with specialized functions. Although the biogenesis of these organelles is well understood at the morphological level, the machinery involved in plant vacuole formation is largely unknown. We have recently identified an Arabidopsis mutant, vcl1, that is deficient in vacuolar formation. VCL1 is homologous to a protein that regulates membrane fusion at the tonoplast in yeast. On the basis of these observations, VCL1 is predicted to play a direct role in vacuolar biogenesis and vesicular trafficking to the vacuole in plants. In this work, we show that VCL1 forms a complex with AtVPS11 and AtVPS33 in vivo. These two proteins are homologues of proteins that have a well-characterized role in membrane fusion at the tonoplast in yeast. VCL1, AtVPS11, and AtVPS33 are membrane-associated and cofractionate with tonoplast and denser endomembrane markers in subcellular fractionation experiments. Consistent with this, VCL1, AtVPS11, and AtVPS33 are found on the tonoplast and the prevacuolar compartment (PVC) by immunoelectron microscopy. We also show that a VCL1-containing complex includes SYP2-type syntaxins and is most likely involved in membrane fusion on both the PVC and tonoplast in vivo. VCL1, AtVPS11, and AtVPS33 are the first components of the vacuolar biogenesis machinery to be identified in plants.
doi:10.1091/mbc.E02-08-0509
PMCID: PMC149977  PMID: 12589039
7.  The Plant Vacuolar Sorting Receptor Atelp Is Involved in Transport of Nh2-Terminal Propeptide-Containing Vacuolar Proteins in Arabidopsis thaliana 
The Journal of Cell Biology  2000;149(7):1335-1344.
Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH2-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.
PMCID: PMC2175142  PMID: 10871276
protein traffic ; Golgi apparatus; COOH-terminal propeptide; plant vacuole barley aleurain
8.  Interactions between Syntaxins Identify at Least Five SNARE Complexes within the Golgi/Prevacuolar System of the Arabidopsis Cell 
Molecular Biology of the Cell  2001;12(12):3733-3743.
The syntaxin family of soluble N-ethyl maleimide sensitive factor adaptor protein receptors (SNAREs) is known to play an important role in the fusion of transport vesicles with specific organelles. Twenty-four syntaxins are encoded in the genome of the model plant Arabidopsis thaliana. These 24 genes are found in 10 gene families and have been reclassified as syntaxins of plants (SYPs). Some of these gene families have been previously characterized, with the SYP2-type syntaxins being found in the prevacuolar compartment (PVC) and the SYP4-type syntaxins on the trans-Golgi network (TGN). Here we report on two previously uncharacterized syntaxin groups. The SYP5 group is encoded by a two-member gene family, whereas SYP61 is a single gene. Both types of syntaxins are localized to multiple compartments of the endomembrane system, including the TGN and the PVC. These two groups of syntaxins form SNARE complexes with each other, and with other Arabidopsis SNAREs. On the TGN, SYP61 forms complexes with the SNARE VTI12 and either SYP41 or SYP42. SYP51 and SYP61 interact with each other and with VTI12, most likely also on the TGN. On the PVC, a SYP5-type syntaxin interacts specifically with a SYP2-type syntaxin, as well as the SNARE VTI11, forming a SNARE complex likely involved in TGN-to-PVC trafficking.
PMCID: PMC60751  PMID: 11739776
9.  The Plant Vesicle-associated SNARE AtVTI1a Likely Mediates Vesicle Transport from the Trans-Golgi Network to the Prevacuolar Compartment 
Molecular Biology of the Cell  1999;10(7):2251-2264.
Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process.
PMCID: PMC25440  PMID: 10397763
10.  AtVPS45 Complex Formation at the trans-Golgi Network 
Molecular Biology of the Cell  2000;11(7):2251-2265.
The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide–sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.
PMCID: PMC14917  PMID: 10888666

Results 1-10 (10)