Targeting and membrane insertion into the endoplasmic reticulum membrane of Saccharomyces cerevisiae essential protein Rot1.
Rot1 is an essential yeast protein that has been related to cell wall biosynthesis, actin cytoskeleton dynamics and protein folding. Rot1 is an N-glycosylated protein anchored to the nuclear envelope-endoplasmic reticulum (ER) membrane by a transmembrane domain at its C-terminal end. Rot1 is translocated to the ER by a post-translational mechanism. Here, we investigate the protein domain required to target and translocate Rot1 to the ER membrane. We found that several deletions of the N-terminal region of Rot1 prevented neither membrane targeting nor the insertion of this protein. Interestingly, we obtained the same results when different truncated forms in the C-terminal transmembrane domain were analyzed, suggesting the presence of an internal topogenic element that is capable of translocating Rot1 to the ER. To identify this sequence, we generated a combination of N- and C-terminal deletion mutants of Rot1 and we investigated their insertion into the membrane. The results show that two regions, amino acids 26-60 and 200-228, are involved in the post-translational translocation of Rot1 across the ER membrane.
Juanes
María Angeles
MA
Departament de Bioquímica i Biologia Molecular, Universitat de València, València, Spain.
Martínez-Garay
Carlos Andrés
CA
Igual
Juan Carlos
JC
Bañó
María Carmen
MC
D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017384:Sequence Deletion
D008565:Membrane Proteins; D018832:Molecular Chaperones; C523667:ROT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins