Research Group Pimpl
-Sorting and Transport of Proteins-
The secretory pathway is the prerequisite for the viability of all eukaryotic cells. This complex biochemical pathway manufactures and distributes proteins, lipids and polysaccharides between intracellular compartments and to the extracellular space. This exceeds by far pure maintenance of independent compartments within the cell, since it is also essential for cell division and cell differentiation as well as cell-cell interactions, permitting the existence of complex multi-cellular organisms, capable of interacting with their environment. Furthermore, the plant secretory pathway not just facilitates plant life and growth under extreme conditions like heat-, drought- and salt-stress, or during plant-pathogen interactions, it also produces and stores proteins as the major food supply for the global population and productive livestock. Our current research is focused on mapping transport routes of vesicular carriers to decipher the dynamic relationship between the intracellular compartments.
Protein sorting in the secretory pathway
A common feature of all eukaryotic cells is the existence of functionally distinct intracellular compartments. These compartments are dynamically interconnected via different vesicular carriers and thus constitute a complex network, termed the secretory pathway (Fig. 1). This pathway manufactures, distributes and degrades macro-molecules like proteins and lipids as a prerequisite for cellular viability and development.
Figure 1: The plant secretory pathway.
Secretion to the plasma membrane (red) leads from the ER via the Golgi and the trans-Golgi network (TGN). ER-resident proteins are retrieved back to the ER (blue). Endocytic transport (black) occurs from the PM to the TGN, the early endosome (EE). The lytic vacuole (LV) (purple) is reached via a prevacuolar compartment (PVC). Transport to the protein storage vacuole (PSV) might occur either from the ER or via dense-vesicles (DV) from the Golgi (grey).
Protein sorting to the lytic vacuole
Sorting of soluble proteins to the lytic vacuole requires vacuolar sorting receptors (VSRs). According to the common view, sorting is initiated by VSR-ligand interactions at the TGN, leading to the formation of a receptor-ligand complex. This complex is sorted into clathrin-coated vesicles, which mediate transport to the prevacuolar compartment (PVC). Upon arrival, the complex dissociates and while the soluble cargo is delivered via fusion of the PVC with vacuole, the receptor is recycled back to sort further ligands.
In yeast and mammalian cells, recycling of vacuolar/lysosomal sorting receptors occurs via a cytosolic protein coat, termed retromer. It consists of a heterotrimeric cargo recognition subunit (VPS35, VPS26 & VPS29) and two proteins of the sorting nexin (SNX) family, which recruit the heterotrimer and drive transport via their C-terminal coiled-coil domains.
To date, very little is known about the sorting and transport mechanisms of plant VSRs.
VSRs recycle from the TGN
In order identify the recycling point of VSRs, we have localized retromer subunits in situ using immunogold electron microscopy. Surprisingly, the sorting nexin SNX2a and the VPS29 subunit of the cargo recognition complex are exclusively localized to the TGN (Fig. 2), indicating that retromer-mediated VSR recycling might occur already at the TGN.
Figure 2: Retromer localizes to the TGN.
IEM localization of the retromer subunits SNX2a (A) and VPS29 (B) in Arabidopsis roots after high-pressure freezing and kryo-substitution (Niemes et al., 2010a).
To analyze retromer-mediated VSR recycling in vivo, we have manipulated retromer function by co-expressing a mutant of SNX2a, lacking the C-terminal coiled-coil domains (SNX2a-<span style="font-family: Symbol;">Δ</span>CC) with the fluorescent VSR-reporter GFP-BP80. The expression of SNX2a-<span style="font-family: Symbol;">Δ</span>CC leads to an accumulation of the VSR reporter GFP-BP80 in close proximity to the Golgi, presumably the TGN (Fig. 3). Since VSR-reporter and endogenous VSRs follow the same transport routes it is likely to assume that endogenous VSRs also accumulate in the presence of the SNX mutant due to perturbed retromer function. However, the vacuolar delivery of the soluble model ligand GFP-sporamin is not affected under these conditions. This demonstrates that post-TGN transport of soluble vacuolar cargo does not require VSRs, which is in agreement with the definition of the TGN as recycling-point of the VSRs.
Figure 3: VSRs are not required for post-TGN sorting of soluble vacuolar cargo.
A, B) Expression of SNX2a-ΔCC accumulates the VSR reporter (PVC marker) GFP-BP80 (green) in close proximity to the Golgi (red), presumably in the TGN. C) In contrast to wortmannin, SNX2a-ΔCC does not perturb vacuolar delivery of the VSR ligand GFP-sporamin as judged by the unchanged presence of the vacuolar degradation product (V) (Niemes et al., 2010a).
VSRs sort cargo in the ER
The identification of the TGN as the recycling point of the VSRs raises the question about the location of the initial receptor-ligand interaction. VSRs and ligands are synthesized in the ER and we wanted to test whether they interact already at this compartment. We therefore generated ER-anchored VSR derivatives by fusing the lumenal ligand binding domain (LBD) of the VSR BP80 to the transmembrane domain (TMD) of the ER-resident chaperone calnexin (CNX) (Figure 4). Coexpression of the ER-anchored receptors with the soluble vacuolar reporter aleurain-GFP results in a strong accumulation of aleurain-GFP in the ER. This ER accumulation is specific for vacuolar cargo and does not generally perturb the ER exit.
Figure 4: Receptor-ligand interaction occurs in the ER.
A) Molecular tools to analyze receptor-ligand interaction in vivo. B, C) BP80-CNX-XFP (green) co-localizes with the ER marker (red) and does not influence ER export of a Golgi marker (red). D, E) BP80-CNX accumulates the vacuolar reporter aleurain-GFP (green) in the ER (Niemes et al.,2010b).
The future aims of the group are (a) to identify the target of the retromer-mediated route and (b) to investigate the dynamics of anterograde and retrograde VSR transport.
Dr. Peter Pimpl
Center for Plant Molecular Biology - ZMBP
University of Tübingen
Auf der Morgenstelle 32
72076 Tübingen, Germany
Phone: +49 (0)7071 - 29 78889
Fax: +49 (0)7071 - 29 5797