The ER luminal C-terminus of AtSec62 is critical for male fertility and plant growth in Arabidopsis thaliana.

Protein translocation into the endoplasmic reticulum (ER) occurs either co- or post-translationally through the Sec translocation system. The Arabidopsis Sec post-translocon is composed of the protein-conducting Sec61 complex, the chaperone-docking protein AtTPR7, the J-domain-containing proteins AtERdj2A/B and the yet uncharacterized AtSec62. Yeast Sec62p is suggested to mainly function in post-translational translocation, whereas mammalian Sec62 also interacts with ribosomes. In Arabidopsis, loss of AtSec62 leads to impaired growth and drastically reduced male fertility indicating the importance of AtSec62 in protein translocation and subsequent secretion in male gametophyte development. Moreover, AtSec62 seems to be divergent in function as compared with yeast Sec62p, since we were not able to complement the thermosensitive yeast mutant sec62-ts. Interestingly, AtSec62 has an additional third transmembrane domain in contrast to its yeast and mammalian counterparts resulting in an altered topology with the C-terminus facing the ER lumen instead of the cytosol. In addition, the AtSec62 C-terminus has proven to be indispensable for AtSec62 function, since a construct lacking the C-terminal region was not able to rescue the mutant phenotype in Arabidopsis. We thus propose that Sec62 acquired a unique topology and function in protein translocation into the ER in plants.

In Vivo Radiolabeling of Arabidopsis Chloroplast Proteins and Separation of Thylakoid Membrane Complexes by Blue Native PAGE.

The investigation of membrane protein complex assembly and degradation is essential to understand cellular protein dynamics. Blue native PAGE provides a powerful tool to analyze the composition and formation of protein complexes. Combined with in vivo radiolabeling, the synthesis and decay of protein complexes can be monitored on a timescale ranging from minutes to several hours. Here, we describe a protocol to analyze thylakoid membrane complexes starting either with (35)S-methionine labeling of intact Arabidopsis leaves to investigate protein complex dynamics or with unlabeled leaf material to monitor steady-state complex composition.

The extreme Albino3 (Alb3) C terminus is required for Alb3 stability and function in Arabidopsis thaliana.

MAIN CONCLUSION: The extreme Alb3 C terminus is important for Alb3 stability in a light dependent manner, but is dispensable for LHCP insertion or D1 synthesis. YidC/Oxa1/Alb3 dependent insertion of membrane proteins is evolutionary conserved among bacteria, mitochondria and chloroplasts. Chloroplasts are challenged by the need to coordinate membrane integration of nuclear encoded, post-translationally targeted proteins into the thylakoids as well as of proteins translated on plastid ribosomes. The pathway facilitating post-translational targeting of the light-harvesting chlorophyll a/b binding proteins involves the chloroplast signal recognition particle, cpSRP54 and cpSRP43, as well as its membrane receptor FtsY and the translocase Alb3. Interaction of cpSRP43 with Alb3 is mediated by the positively charged, stromal exposed C terminus of Alb3. In this study, we utilized an Alb3 T-DNA insertion mutant in Arabidopsis thaliana lacking the last 75 amino acids to elucidate the function of this domain (alb3∆C). However, the truncated Alb3 protein (Alb3∆C) proved to be unstable under standard growth conditions, resulting in a reduction of Alb3∆C to 20 % of wild-type levels. In contrast, accumulation of Alb3∆C was comparable to wild type under low light growth conditions. Alb3∆C mutants grown under low light conditions were only slightly paler than wild type, accumulated almost wild-type levels of light harvesting proteins and were not affected in D1 synthesis, therefore showing that the extreme Alb3 C terminus is dispensable for both, co- and post-translational, protein insertion into the thylakoid membrane. However, reduction of Alb3∆C levels as observed under standard growth conditions resulted not only in a severely diminished accumulation of all thylakoid complexes but also in a strong defect in D1 synthesis and membrane insertion.