Targeting and membrane-insertion of a sunflower oleosin in vitro and in Saccharomyces cerevisiae: the central hydrophobic domain contains more than one signal sequence, and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism.
A range of N- and C-terminal deletions of an oleosin from Helianthus annuus L. were used to study the endoplasmic reticulum (ER) targeting and membrane insertion of this protein both in vitro and in vivo in yeast ( Saccharomyces cerevisiae). Neither the N- nor the C-terminal hydrophilic domains are important for targeting and/or membrane insertion, with all the information required for these processes located within the central hydrophobic region of the protein. However, in vitro membrane-insertion experiments suggest that these domains are important for a correct topology of the oleosin within the ER membrane. The first half of the hydrophobic central domain, flanked by the positively charged N-terminal domain, is likely to function as a type-II signal-anchor (SAII) sequence. However, in the absence of the N-terminal 26 residues of this domain, the proline-knot region and the second half of this hydrophobic domain are sufficient to direct oleosin to the ER and to allow stable (but far less efficient) integration of the protein into the membrane. Taken together, these results indicate that oleosin contains more than one domain that is capable of interacting with the signal recognition particle to direct the protein to the ER membrane.
Beaudoin
Frédéric
F
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, UK. frederic.beaudoin@bbsrc.ac.uk
Napier
Johnathan A
JA
D000595:Amino Acid Sequence; D001692:Biological Transport; D002458:Cell Fractionation; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005786:Gene Expression Regulation; D006368:Helianthus / Q000235:genetics* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
D008565:Membrane Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; C079350:oleosin, Helianthus annuus