Characterizing the molecular function of plastoglobules

Plastoglobules (PGs) are lipoprotein particles found in chloroplasts.
They are rich in non-polar lipids (triacylglycerols, esters) as well as prenylquinones, plastoquinone and tocopherols.
Plastoglobules are often associated with thylakoid membranes, suggesting lipid exchange with thylakoids.

Ultrastructure of anArabidopsis thaliana chloroplast (left) and visualization of the structure of a plastoglobule (PG, right) by electron microscopy.

Proteins called plastoglobulins (PGL) or PAP/fibrillins are associated with the stromal surface of plastoglobules.
These proteins probably regulate the structure of plastoglobules and are very abundant, particularly in chromoplasts.

Model of a plastoglobule structure

We use molecular genetics and biochemical techniques to study the function and targeting of these proteins.

Protoplast expressing a fusion between pGL-YFP (green) and – CFP (blue) under confocal fluorescence microscopy. Red represents chlorophyll auto-fluorescence.

Physiological factors such as senescence and environmental stress lead to an increase in the size and number of plastoglobules. However, the molecular function of plastoglobules in these processes is not known.

To understand the molecular functions of plastoglobulins, we used the Arabidopsis model system and analyzed the plastoglobulin proteome. In addition to plastoglobulins, we identified enzymes involved in sugar and lipid metabolism (Vidi et al., 2006).

In particular, the enzyme tocopherol cyclase (VTE1) has been identified as a component of the plastoglobule. In addition, plastoglobules have been shown to be the main site of tocopherol accumulation in Arabidopsis.

The final destination of tocopherol is most likely the thylakoid membrane, where it is thought to prevent the oxidative degradation of fatty acids by trapping reactive oxygen species. The presence of TEV1 and tocopherols, combined with an increase in the size and number of plastoglobules, provides a molecular explanation for the role of plastoglobules under conditions of oxidative stress.

TIC and TOC – or how proteins enter chloroplasts

Light triggers a developmental program in plants that makes them photoautotrophic. A key step in this process is the biogenesis of chloroplasts from undifferentiated proplastids. The assembly of the photosynthetic apparatus requires the import of around 2,000 different nuclear-encoded proteins. The nuclear-encoded proteins are synthesized in the cytosol as precursors with an N-terminal targeting peptide (transit peptide) and must be imported into the nascent chloroplast.

Development of etioplasts (left) into chloroplasts (right).
Photo: T. Kleffmann (Institute of Plant Sciences ETH Zürich)

The chloroplast is surrounded by an envelope consisting of two membranes. Both membranes contain translocons that facilitate the import of precursor proteins. These translocons are called Toc (chloroplast outer membrane translocon) and Tic (chloroplast inner membrane translocon) complexes. The Toc complex is made up of three main components forming a stable complex. Toc159 (number indicates molecular mass in kD) and Toc34 are surface-exposed, GTP-binding integral membrane proteins. Both proteins share a highly conserved GTP-binding domain. Available data indicate that the two proteins act in concert to recognize the chloroplast-targeted peptide. Toc159 is thought to act as a primary receptor for transit sequences. An alternative model in which Toc34 functions as a primary receptor and Toc159 as a GTP-dependent translocation motor has also been proposed. Toc75, the third component, forms at least part of a hydrophilic channel through which precursors are translocated across the outer membrane. Work in our laboratory aims to elucidate the function of the Toc-GTPase using a combination of in vivo and in vitro methods.

Protein import model in chloroplasts. In blue, the cytosol. In white, the chloroplast envelope. Green: stroma.

Evidence accumulated by a number of groups suggests the following model for the chloroplast protein import machinery: the precursor protein is synthesized in the cytosol with a transit peptide at the N-terminus. In a first energy-independent step, the precursor binds to Toc159. In a first energy-independent step, the precursor binds to Toc159. In a second step requiring ATP and GTP, the precursor crosses the outer membrane and encounters the components of the Tic complex on the inner membrane surface. In a third step requiring ATP, the precursor crosses both membranes simultaneously.

What is the physiological significance of the components of the chloroplast protein import machinery?

We have recently isolated an Arabidopsis mutant, called ppi2 (plastid protein import mutant 2), which lacks atToc159. Compared with wild-type chloroplasts, ppi2 plastids resemble undifferentiated proplastids lacking thylakoid membranes and starch granules.

Phenotype of theArabidopsis thaliana ppi2 mutant

We analyzed the distribution of atToc159 between the soluble and chloroplastic fractions. To our great surprise, atToc159 was present in both fractions. Transient expression of a GFP fusion in Arabidopsis protoplasts confirms this result. We can see that the GFP fusion is concentrated on the surface of the chloroplast, but is also present in the cytosol surrounding the vacuole.

Soluble and outer membrane forms of Toc159.

The data show that atToc159 exists in both soluble and membrane-bound forms. We hypothesize that atToc159 can switch from one form to the other, and that this cycle may be controlled by GTP.

Model for the initiation of precursor import at the chloroplast outer membrane.

The Tic110, -62, -55, -40, -22 and -20 proteins were identified as components of the import machinery at the inner envelope membrane. Tic110 was found in association with a late translocation intermediate, spanning both envelope membranes, suggesting a role in the terminal stages of import. The Tic55 and Tic62 proteins were co-isolated with the Tic110 protein in a complex. Both proteins contain redox elements, suggesting that protein import may be controlled by the redox potential of chloroplasts. Finally, Tic20 and Tic22 cross-linked to precursor proteins stably inserted across the chloroplast outer membrane, but not yet having reached the chloroplast stroma. These data suggest that Tic20 and Tic22 initiate translocation of the incoming precursor across the inner membrane. In vivo analysis of the Arabidopsis ortholog of Tic20, atTic20, demonstrates that this protein plays a role in pre-protein translocation across the inner envelope membrane.

Although Tic110 is a widely recognized component of the import machinery, its topology and function are controversial. Tic110 is an integral membrane protein anchored by a transmembrane domain composed of two predicted transmembrane helices near the N-terminus of the protein. The C-terminal end, which is large and around 90 kD, is thought to be largely a-helical and to contain no hydrophobic membrane-anchoring a-helices. Conflicting reports have been published concerning the topology of Tic110, concluding that the C-terminal domain extends either into the stroma or into the intermembrane space between the two envelope membranes. Reported interactions with the stromal chaperones, cpn60 and ClpC, suggest that Tic110 may function to couple protein import to protein folding. In addition, it has been proposed that ClpC acts as a motor for protein transport across the inner membrane. Another function of Tic110 has also been proposed. Recombinant Tic110, incorporated into lipid bilayers, showed a channel activity influenced by precursor proteins. The channel function is attributed to the C-terminal domain, suggesting that it contains transmembrane segments. Our current research focuses on the in vivo and biochemical function of atTic110.