0000000000008079
AUTHOR
Carlos A. Martínez-garay
Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit
Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.
Karyopherin Msn5 is involved in a novel mechanism controlling the cellular level of cell cycle regulators Cln2 and Swi5
ABSTRACT The yeast β-karyopherin Msn5 controls the SBF cell-cycle transcription factor, responsible for the periodic expression of CLN2 cyclin gene at G1/S, and the nuclear export of Cln2 protein. Here we show that Msn5 regulates Cln2 by an additional mechanism. Inactivation of Msn5 causes a severe reduction in the cellular content of Cln2. This occurs by a post-transcriptional mechanism, since CLN2 mRNA level is not importantly affected in asynchronous cultures. Cln2 stability is not significantly altered in msn5 cells and inactivation of Msn5 causes a reduction in protein level even when Cln2 is stabilized. Therefore, the reduced amount of Cln2 in msn5 cells is mainly due not to a higher …
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 dom…
A transmembrane serine residue in the Rot1 protein is essential for yeast cell viability
Polar residues are present in TM (transmembrane) helices and may influence the folding or association of membrane proteins. In the present study, we use an in vivo approach to analyse the functional and structural roles for amino acids in membrane-spanning motifs using the Rot1 (reversal of Tor2 lethality 1) protein as a model. Rot1 is an essential membrane protein in Saccharomyces cerevisiae and it contains a single TM domain. An alanine insertion scanning analysis of this TM helix revealed that the integrity of the central domain is essential for protein function. We identified a critical serine residue inside the helix that plays an essential role in maintaining cell viability in S. cere…