Current Projects

Our research focuses on the regulation of protein and membrane trafficking mechanisms in plant cells. Our goal is to understand how cells control the flow of proteins between different cellular compartments. We are working on four projects:

Endosomal sorting of membrane proteins for degradation:

Cells are able to interact with other cells and their environment through molecules located in their surface and therefore, it is critical for a cell to be able to control the protein composition of its plasma membrane. We are analyzing the mechanisms that mediate the recognition and degradation of plasma membrane proteins in plants. These mechanisms allow cells to control theabundance of key plasma membrane proteins involved in cell signaling, growth, and development, such as activated receptors, hormone transporters, and ion channels. The degradation of plasma membrane proteins is mediated by membrane-bound organelles called endosomes. Plasma membrane proteins internalized by endocytosis are sorted for degradation in endosomes called multivesicular bodies (MVBs).
We are focusing on understanding how endocytosed plasma membrane proteins, such as activated receptors, auxin transporters, and ion channels, are recognized in endosomes and sorted for degradation. Soluble proteins that are not anchored into membranes can be degraded by cytoplasmic proteases that belong to the 26S-proteasome degradation pathway. But how does a cell degrade a protein that is inserted in the plasma membrane? Most plasma membrane proteins are flagged for degradation at the plasma membrane by conjugation of an ubiquitin molecule to specific lysine residues on the target protein. In many organisms, ubiquitination is enough to trigger internalization of plasma membrane proteins and their subsequent delivery to endosomes. Endosomal protein complexes are then able to recognize ubiquitinated membrane proteins and sort them into intraluminal vesicles, giving rise to MVBs. When MVBs fuse with lysosomes/vacuoles, the endosomal vesicles are released in the vacuolar lumen and degraded.

The endosomal recognition of ubiquitinated cargo and formation of intraluminal vesicles is mediated by the coordinated action of more than 30 different proteins. Most of these proteins are organized in complexes called ESCRT-0, -I, -II, and –III (Endosomal Sorting Complex Required for Transport)[15, 31, 34]. ESCRT-0 recognizes, selects, and clusters the ubiquitinated membrane proteins on the endosomal membrane; ESCRT-I and ESCRT-II induce membrane deformation and localize to the neck of the nascent vesicle; ESCRT-III together with an ATPase called SKD1/Vps4 are important for membrane scission and release of the nascent vesicle into the MVB lumen. We are analyzing the function of ESCRT component in endosomal sorting and plant development.

mvb pathway
Diagram of endosomal trafficking pathways in plant cells. Plasma membrane proteins are synthesized in the endosplasmic reticulum (ER), transported through the Golgi/TGN, and delivered to the plasma membrane. At the plasma membrane, some proteins (for example activated receptors) can be ubiquitinated, internalized into the cell by endocytosis, and delivered to endosomes. At endosomes, the ubiquitinated plasma membrane proteins are recognized by endosomal protein complexes and sorted into internal vesicles that are subsequently degraded in vacuoles. Upper left corner, electron micrograph of a plant MVB.

Delivery of proteins to the vacuole:

Another research area in my laboratory is the transport and post-translational processing of storage proteins in different cell types of the corn kernel. We have found that the same storage proteins undergo different trafficking pathways in different cell types of the corn endosperm (the tissue that stores most of the starch and proteins found in the corn kernel). This has very important implications in agriculture since the trafficking pathways and cellular accumulation sites have drastic effects on post-translational modifications of storage proteins, affecting their antigenicity and digestibility for human and livestock consumption, kernel texture, and yield. We are monitoring the trafficking pathways of these proteins using live-cell imaging; to this end we have developed a method for generating transgenic endosperms in vitro. In addition, we have performed extensive electron tomography analysis of endosperm cells. We have found that a novel, ATG8-independent autophagic mechanism mediates the delivery of storage proteins to vacuoles in endosperm cells (Reyes et al., The Plant Cell in press).

 aleurone

Storage protein transport in maize aleurone cells: Electron tomographic reconstruction of a developing maize aleurone cell containing vacuoles with large aggregates of storage proteins (red) and intravacuolar membranes (green). Mitochondria (gold), plastids (green), lipid bodies (blue), and ribosomes (grey) are abundant in the cytoplasm.

Trafficking of cell wall components during the differentiation of secondary cell walls:

Another area of interest in my laboratory is the identification of proteins required for polysaccharide and lignin synthesis and transport during the formation of secondary cell walls in Arabidopsis and maize. This is a collaborative project with Sebastian Bednarek (Department of Biochemistry) and Patrick Masson (Department of Genetics) supported by the Great Lakes Bioenergy Research Center (US Department of Energy), which is devoted to biofuel research. We are identifying and characterizing new genes affecting secondary wall formation in plants. We have optimized an in vitro system that allows us to induce differentiation of secondary cell walls in Arabidopsis cultured cells. We are isolating membrane fractions of these cells to perform proteomics analysis and identify novel factors involved in secondary cell wall formation. We are analyzing genes that control lignin deposition and affect cell wall structure and biomass digestibility for ethanol production.

 

Trafficking of anthocyanins:

 

In contrast to the extensive knowledge available on plant metabolism, very little is known on how plants transport often toxic or highly reactive chemicals from their site of synthesis to where they are ultimately stored. Proper trafficking and storage of plant compounds (phytochemicals) is often a bottleneck in efforts aimed at the rationale engineering of plant metabolism. Thus, understanding the cellular and molecular mechanisms involved in phytochemical trafficking is of the utmost significance. This a collaborative project with Erich Grotewold (Ohio State University) to analyze the subcellular compartmentalization of anthocyanin biosynthesis and storage.


Experimental Approaches

We use a variety of multidisciplinary techniques, including cryofixation/freeze substitution, electron and confocal microscopy, dual-axis electron tomography, immunogold-labeling, subcellular fractionation, RT-PCR and qPCR, expression of fluorescent fusion proteins in Arabidopsis thaliana, tobacco BY2 cells, maize, and yeast, and production of recombinant proteins in bacteria for biochemical studies.

Golgi Tomographic Reconstruction 1        Golgi Tomographic Reconstruction 2

Tomographic 3D reconstructions of Golgi in an Arabidopsis thaliana embryo cell