Showing papers by "Natasha V. Raikhel published in 2012"

Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis

Abstract: The endomembrane system is a complex and dynamic intracellular trafficking network. It is very challenging to track individual vesicles and their cargos in real time; however, affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified. Pioneering this approach in plants, we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles. The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment. We identified a complete SYP61 SNARE complex, including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta. We further identified the SYP121-complex and cellulose synthases, suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane. The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos. The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks. The approach is widely applicable and can afford the study of several vesicle populations in plants, which can be compared with the SYP61 vesicle proteome.

Arabidopsis ribosomal proteins control developmental programs through translational regulation of auxin response factors

Abstract: Upstream ORFs are elements found in the 5′-leader sequences of specific mRNAs that modulate the translation of downstream ORFs encoding major gene products. In Arabidopsis, the translational control of auxin response factors (ARFs) by upstream ORFs has been proposed as a regulatory mechanism required to respond properly to complex auxin-signaling inputs. In this study, we identify and characterize the aberrant auxin responses in specific ribosomal protein mutants in which multiple ARF transcription factors are simultaneously repressed at the translational level. This characteristic lends itself to the use of these mutants as genetic tools to bypass the genetic redundancy among members of the ARF family in Arabidopsis. Using this approach, we were able to assign unique functions for ARF2, ARF3, and ARF6 in plant development.

Small molecules present large opportunities in plant biology.

Abstract: Since the introduction of chemical genomics to plant biology as a tool for basic research, the field has advanced significantly. There are now examples of important basic discoveries that demonstrate the power and untapped potential of this approach. Given the combination of protein and small-molecule complexity, new phenotypes can be described through the perturbation of cellular functions that can be linked to growth and developmental phenotypes. There are now clear examples of overcoming functional redundancy in plants to dissect molecular mechanisms or critical pathways such as hormone signaling and dynamic intracellular processes. Owing to ongoing advances, including more sophisticated high-content screening and rapid approaches for target identification, the field is beginning to move forward. However, there are also challenges to improve automation, imaging, and analysis and provide chemical biology resources to the broader plant biology community.

Discovery of new modules in metabolic biology using ChemoMetabolomics.

Abstract: The use of small molecules to transiently modulate protein function can circumvent the limitations of classical genetic approaches caused by gene redundancy and lethality. Although chemical genomics and genetics screens facilitate the characterization of new biological components, they have infrequently led to the identification of target proteins involved in metabolism. The current state of metabolomics technologies permits the detection of thousands of molecules, allowing the exploration of yet uncharacterized metabolic pathways. The combination of these two approaches, termed here “ChemoMetabolomics”, is a promising application of both technologies. This novel approach will facilitate the detection of metabolic modulators, the dissection of the crosstalk in the metabolic network, and the development of hypotheses on how changes in metabolism affect developmental or cellular responses. Furthermore, it will facilitate the elucidation of the linkage between metabolic and developmental programs and assist the gain of an elaborated view of biological processes at the system level.

Chemical Effectors of Plant Endocytosis and Endomembrane Trafficking

Abstract: Plant endocytosis and endomembrane trafficking relies on the coordination of a highly organized and dynamic network of intracellular organelles. Membrane trafficking and associated signal transduction pathways provide critical cellular regulation of plant development and response to environmental stimuli. However, the efficiency of studies on this complex network has been hampered due to the rapid and dynamic nature of endomembrane trafficking as well as gene redundancy and embryonic lethality in mutagenesis-based strategies. Chemical genomics emerged in recent years as a complementary approach to illuminate biological functions through the integration of organic chemistry, biology, and bioinformatics to overcome gene redundancy. The approach presents significant advantages in dosage dependence and reversibility, which offers the ideal ability to study dynamic endomembrane trafficking processes in real time. In this chapter, several successful examples of chemical screening focused on the endomembrane system is presented to illuminate the efficiency and power of chemical genomics in dissecting endomembrane trafficking and its regulation of plant development and environmental responses. Perspectives are also presented to suggest directions for future development of this field.