Decoding the Divergent Subcellular Location of Two Highly Similar Paralogous LEA Proteins

Abstract

Many mitochondrial proteins are synthesized as precursors in the cytosol with an N-terminal mitochondrial targeting sequence (MTS) which is cleaved off upon import. Although much is known about import mechanisms and MTS structural features, the variability of MTS still hampers robust sub-cellular software predictions. Here, we took advantage of two paralogous late embryogenesis abundant proteins (LEA) from Arabidopsis with different subcellular locations to investigate structural determinants of mitochondrial import and gain insight into the evolution of the LEA genes. LEA38 and LEA2 are short proteins of the LEA_3 family, which are very similar along their whole sequence, but LEA38 is targeted to mitochondria while LEA2 is cytosolic. Differences in the N-terminal protein sequences were used to generate a series of mutated LEA2 which were expressed as GFP-fusion proteins in leaf protoplasts. By combining three types of mutation (substitution, charge inversion, and segment replacement), we were able to redirect the mutated LEA2 to mitochondria. Analysis of the effect of the mutations and determination of the LEA38 MTS cleavage site highlighted important structural features within and beyond the MTS. Overall, these results provide an explanation for the likely loss of mitochondrial location after duplication of the ancestral gene.

Structural properties and cellular expression of AfrLEA6, a group 6 late embryogenesis abundant protein from embryos of Artemia franciscana

“…Similar tardigrade-specific intrinsically disordered proteins have also been described . Within these organisms, LEA proteins are present across a range of subcellular compartments including the nucleus, endoplasmic reticulum, Golgi apparatus, mitochondrion, and chloroplast (Avelange-Macherel et al 2018;Boswell and Hand 2014;Hand et al 2011;Tripathi et al 2012;Tunnacliffe et al 2010;Tunnacliffe and Wise 2007). Classification schemes place them into six groups, of which A. franciscana uniquely expresses LEA proteins from three different groups (1, 3, and 6; Hand and Menze 2015; Tunnacliffe and Wise 2007;Warner et al 2010Warner et al , 2012.…”

The in vitro structure and functions of the disordered late embryogenesis abundant three proteins

“…The arrows indicate the putative cut‐that would result in the removal of the N‐terminal sequence. Note that studies have shown that AtLEA3‐3 does not locate to the mitochondrion, but is found in the cytoplasm and the nucleus 37,44 . However, the full‐length AtLEA3‐3 protein could not be expressed in soluble form for biochemical characterization (data not shown), so we therefore truncated the AtLEA3‐3 gene at the position equivalent to residue 29, although this MTS cleavage site may not be cut in vivo 44 .…”

CropPAL for discovering divergence in protein subcellular location in crops to support strategies for molecular crop breeding

“…In fact, our data showed that about 10% of genes produce ASVs with products targeted to different compartments. The fact that these are more likely preserved after duplication supports the relevance of subcellularly diverse ASVs (Avelange-Macherel et al, 2018;Qiu et al, 2019). Changes in ASV expression are somewhat explored at the RNA level but poorly understood at the protein level (Zhang et al, 2019).…”