Experimental determination of organelle targeting-peptide cleavage sites using transient expression of green fluorescent protein translational fusions

The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress

“…Table 3 displays the targeting peptide cleavage sites we were able to determine for LEA proteins identified in plastids or mitochondria. The cleavage sites for LEA23-GFP and LEA24-GFP, which are localized in plastids, were determined previously (Candat et al, 2013). In the case of LEA11 and LEA12, which are two paralogous proteins targeted to plastids (Figure 4), the cleavage site could be only be determined for LEA11-GFP (Table 3).…”

“…To facilitate the proper structural and functional characterization of organellar proteins, it is critical to determine the targeting peptide cleavage sites. We previously described a method to experimentally determine the targeting peptide cleavage site of C-terminal GFP fusion proteins imported into mitochondria or chloroplasts during transient expression in protoplasts (Candat et al, 2013). In this approach, protoplasts expressing LEA-GFP proteins are lysed and fusion proteins are immune-captured with GFP antibodies bound to magnetic microbeads.…”

“…However, from the examination of the published images of Arabidopsis RESPONSIVE TO AB-SCISIC ACID28 (Atrab28 and LEA31) stably expressed as a GFP fusion in Arabidopsis (Borrell et al, 2002), the fusion protein appears in fact to be distributed in both the cytosol and nucleus. (Candat et al, 2013).…”

A mechanistic model of COR15 protein function in plant freezing tolerance: integration of structural and functional characteristics

“…[1][2][3] Constitutive expression of COR15A 4 or COR15B resulted in increased freezing tolerance in non-acclimated Arabidopsis plants. Moreover, simultaneous knock-down of both corresponding genes specifically reduced freezing tolerance after cold acclimation, indicating the necessity of the COR15 proteins to attain full cold acclimation.…”

Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo

“…The genes are localized in the nuclear genome, where they are arranged as a tandem repeat. The encoded proteins are targeted to the chloroplast stroma via signal peptides (Lin and Thomashow, 1992b;Nakayama et al, 2007;Candat et al, 2013) resulting in mature proteins of about 9 kD. Both mature proteins are highly hydrophilic and predominantly unstructured in solution, whereas they fold into amphipathic a-helices during drying.…”