Cis-acting sequences involved in targeting Trm1p-II to the inner nuclear membrane in Saccharomyces cerevisiae
Open Access
- Author:
- Stauffer, Karen A
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 28, 2006
- Committee Members:
- Anita Klein Hopper, Committee Chair/Co-Chair
Kristin Ann Eckert, Committee Chair/Co-Chair
Hui Ling Chiang, Committee Member
Richard John Frisque, Committee Member
Leslie Joan Parent, Committee Member - Keywords:
- protein targeting
peripheral protein
nucleus
inner nuclear membrane
tRNA modification
yeast - Abstract:
- The mechanism of targeting a peripheral protein to the inner nuclear membrane (INM) is not understood. Sequences of Trm1p-II, a peripheral INM protein in Saccharomyces cerevisiae, were examined to define the cis-acting regions mediating targeting. Both a 9-amino acid nuclear localization sequence (NLS) targeting -galactosidase to the nuclear interior as well as a 60-amino acid membrane targeting sequence directing -galactosidase to the INM were uncovered. Trm1p-II may possess two NLSs; a classical basic NLS at amino acids 95-102 (NLS#1) and a putative NLS at amino acids 544-552 (NLS#2). NLS#1 is likely recognized by the classical importin, importin , based on amino acid sequence similarities with known importin  substrates, while NLS#2 is recognized by the non-classical importers, Nmd5p and Kap120p. Several regions of Trm1p-II are necessary for INM targeting, including the N-terminus and two internal regions, as deleting these regions releases Trm1-II-GFP from the INM into the nucleoplasm. However, a single cis-acting sequence of Trm1p-II (amino acids 89-151) is sufficient to target passenger protein(s) to the INM. Competition experiments suggest Trm1p-II may be tethered to a second nuclear component, functioning in addition to the membrane partner, to retain Trm1p-II within the nucleus. Ice2p was identified in a genome-wide screen for mutants that mislocate Trm1-II-GFP, suggesting Ice2p may have a role in targeting of Trm1p-II to the INM; deletion of ICE2 releases Trm1-II-GFP from the INM into the nucleoplasm. However, a -galactosidase fusion protein containing amino acids 73-151 of Trm1p-II is maintained at the INM in ice2 cells suggesting Ice2p may have a role in the distribution of Trm1p-II around the INM (possibly by regulating a protein(s) involved in this process) rather than functioning directly by binding Trm1p-II at the membrane. As Trm1p-II is acetylated at its N-terminus, we propose a bipartite model for targeting to the INM that requires this protein modification to expose a cis-acting sequence within amino acids 89-151 for binding Trm1p-II to its INM-binding partner. We believe Trm1p-II is targeted to a specific site at the INM and subsequently spread from this site, accounting for the variations in nuclear localization observed with different Trm1p-II fusion proteins.