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First published online 27 May 2003
doi: 10.1242/jcs.00597

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Protein translocation across the endoplasmic reticulum membrane in cold-adapted organisms

Karin Römisch1,*, Nicola Collie1, Nelyn Soto2, James Logue2, Margaret Lindsay1, Wiep Scheper1 and Chi-Hing C. Cheng2

1 University of Cambridge, CIMR and Department of Clinical Biochemistry, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK
2 Department of Animal Biology, University of Illinois at Urbana-Champaign, IL 61801, USA



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  		Fig. 1. Protein translocation into the ER of the Antarctic yeast C. adeliensis and the mesophilic yeast S. cerevisiae. Upper: limiting amounts of microsomal protein were incubated with excess radiolabelled secretory precursor (p{Delta}gp{alpha}f) for 0 (lanes 1, 5) or 15 minutes (all other lanes) at the indicated temperatures and samples analyzed by SDS-PAGE. Signal-cleavage ({Delta}gp{alpha}f) was used as a measure of translocation and was quantified using a phosphorimager. Signal-cleaved {Delta}gp{alpha}f was >90% protease protected. Samples were taken in duplicate, and the experiment repeated twice. The difference between translocation at different temperatures is indicated (x). Lower: translocation at 20°C for each species was set to 100% to illustrate the relative decrease in translocation efficiency with decreasing temperature.

 


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  		Fig. 2. Electron micrographs of Antarctic and mesophilic Cryptococci. Upper: Cryptococcus antarcticus. The large mitochondria were typical for this species; they may provide a respiratory advantage at the extremely low habitat temperature of this organism. Middle: Cryptococcus adeliensis. Lower: Cryptococcus laurentii. Bar, 1 µm. Mitochondria (m) and ER (arrows) are indicated.

 


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  		Fig. 3. Amino-acid sequences of Sec61{alpha} from mouse, frog, temperate and cold-water fishes. Species are indicated on the left, Genbank accession number of sequences in brackets: Mm Mus musculus (NP058602), Dr Danio rerio (AAK40295), Fr Fugu rubripes (http://fugu.hgmp.mrc.ac.uk), Om Onchorhynchus mykiss (AAK29081), Sr Sea Raven/Hemitripterus americanus (AY103473), Na Notothenia angustata (AY103472), Pb Pagothenia borchgrevinki (AY103471), Ha Harpagifer antarcticus (AY113840), Dm Dissostichus mawsoni (AY113841), Go Gadus ogac (AY103475), Bs Boreogadus saida (AY103474). Sequence alignments were performed using Multalin. Identical residues are indicated by periods. Transmembrane regions are boxed.

 


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  		Fig. 4. Protein translocation into ER membranes of the Antarctic fish D. mawsoni, carp and dog. (A) Post-translational import of the truncated secretory precursor PPL86 into limiting amounts of microsomes from D. mawsoni (black), carp (grey) and dog (light grey) was assayed as described in the Materials and Methods section at the indicated temperatures. Samples were taken in duplicate and the experiment repeated twice. Translocation was defined as the percentage of signal-cleaved PL56 associated with microsomes. (B) Quantitative immunoblot of Sec61{alpha}. The indicated amounts of microsomal protein were resolved on a 12.5% gel, and Sec61{alpha} was detected with polyclonal antibodies against the dog protein and [125I]-Protein A.

 


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  		Fig. 5. Lipid composition and fluidity of liver microsomes from four vertebrate species. (A) Correlation between unsaturated fatty acid content and body temperature. (B) 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy at 21°C for each species (mean±s.d., three separate preparations). Note that the relationship between DPH anisotropy values for different species does not change significantly when measurements are done over a temperature range between 2 and 40°C (Logue et al., 2000Go).

 


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  		Fig. 6. Sequence changes in the loop between transmembrane domains 7 and 8 of Sec61{alpha} may improve protein translocation across the ER membrane at low temperature. (A) Sequences of the loop regions from mouse, all fishes and cold-water fishes. Positions of mutations in yeast Sec61p defective in translocation are indicated by asterisks. (B) Sec61{gamma} sequences from M. musculus, X. laevis and H. antarticus (AY258259). The transmembrane region is boxed. (C) Adaptations to the cold may indicate that hinge regions (black circles) in the loop are required for a conformational change during protein translocation into the ER.

 

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© The Company of Biologists Ltd 2003