Regulation of cell cycle transcription factor Swi5 by karyopherin Msn5.

Inactivation of S. cerevisiae ß-karyopherin Msn5 causes hypersensitivity to the overexpression of mitotic cyclin Clb2 and aggravates growth defects of many mutant strains in mitotic exit, suggesting a connection between Msn5 and mitotic exit. We determined that Msn5 controlled subcellular localization of the mitotic exit transcription factor Swi5, since it was required for Swi5 nuclear export. Msn5 physically interacted with the N-terminal end of Swi5. Inactivation of Msn5 caused a severe reduction in cellular levels of Swi5 protein. This effect occurred by a post-transcriptional mechanism, since SWI5 mRNA levels were not affected. The reduced amount of Swi5 in msn5 mutant cells was not due to an increased protein degradation rate, but to a defect in Swi5 synthesis. Despite the change in localization and protein level, Swi5-regulated transcription was not defective in the msn5 mutant strain. However, a high level of Swi5 was toxic in the absence of Msn5. This deleterious effect was eliminated when Swi5 nuclear import was abrogated, suggesting that nuclear export by Msn5 is important for cell physiology, because it prevents toxic Swi5 nuclear accumulation.

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.

Size-exclusion high-performance liquid chromatography in the study of the autoassociating antibiotic gramicidin A in micellar milieu.

Gramicidin A (gA) is a polypeptide antibiotic which forms dimeric channels specific for monovalent cations in biological membranes. It is a polymorphic molecule that adopts several different conformations, double-stranded (ds) helical dimers (pore conformation) and single-stranded beta-helical dimers (channel conformation). This study investigated the conformational adaptability of gramicidin A when incorporated into micelles as membrane-mimetic model system. Taking advantage of our reported, versatile, size-exclusion high-performance liquid chromatography (SE-HPLC) strategy that allows the separation of double-stranded dimers and monomers, we have quantitatively characterized the conformational transition undergone by the peptide in the micellar milieu. The importance of both hydrophobic/hydrophilic moieties of the amphipaths in the stabilization of concrete conformational species is demonstrated using detergents with different hydrocarbon chain length and/or polar head. SE-HPLC is a valuable, rapid, accurate technique for the structural characterization of hydrophobic autoassociating peptides that work in lipid environments such as biological membranes.