Identification of cis-acting sequences and trans-acting factors for targeting the peripherally associated membrane protein, Trm1-II, to the inner nuclear membrane
Year: 2012
Type: article
Abstract: Appropriate nuclear membrane structure is important in eukaryotic cells.Significantly, many human diseases are caused by failure to correctly localize inner nuclear membrane (INM) proteins to the nuclear rim.To gain an understanding of the targeting mechanism of peripherally associated INM proteins, I employed the budding yeast Saccharomyces cerevisiae as a model system and Trm1--II, a tRNA modification enzyme that is peripherally bound to the INM, as a reporter.In Saccharomyces cerevisiae, the TRM1 gene codes for the tRNA--specific N2, N2-dimethylguanosine methyltransferase.It is located in mitochondria and nuclei by encoding two isomeric forms of the proteins, Trm1--I and Trm1--II, via alternative translation initiation.The longer form, Trm1--I, is exclusively localized to the mitochondria while the shorter form, Trm1--II, is located in the nucleus (90%) and mitochondria (10%).The nuclear pool of Trm1--II is peripherally associated with the inner nuclear membrane (INM).The mechanism of targeting Trm1--II to the INM is unclear.To characterize the amino acids important for targeting Trm1--II to the INM, I employed random and site--directed mutagenesis, and uncovered specific amino acids necessary for Trm1--II to locate at the INM.I defined a sequence of ~20 amino acids that contains information necessary to target Trm1--II to the INM.To address whether the newly defined region necessary for Trm1--II INM location is also iii sufficient to target reporter proteins to the INM, I demonstrated that this short peptide causes the redistribution of reporter proteins from the nucleoplasm to the INM.Thus, I identified the first motif for targeting peripherally associated proteins to the INM in yeast (Lai et al., 2009).A genome--wide study that identified subunits of the N--terminal acetyltransferase C (Nat C): Mak3, Mak10, and Mak31 function in Trm1--II INM targeting and suggested N--terminal acetylation of Trm1--II is necessary for it INM location (Murthi and Hopper, 2005).The data suggest that Trm1--II contains information for INM targeting other than the INM targeting motif.To further address the mechanism of Trm1--II INM location, Trm1--II, non--functional INM motif Trm1--II mutant and Trm1--II from cells without Nat C activity were C--terminally tagged with ZZ domain of Protein A (ZZ) or GFP--ZZ to search for Trm1--II interacting partner(s) at the INM using biochemical approaches.Surprisingly, no specific interacting proteins were uncovered by these studies.Rather, the evidence showed that Trm1--II may interact with lipids via the INM targeting motif and associate with the INM.In addition, I identified that without acetylation at N--terminus Trm1--II has the ability to modify tRNAs and to interact with membrane lipids.The data support a model that the INM targeting motif, rather than N--acetylation, is important for Trm1--II INM targeting by facilitating Trm1--II--lipid interaction at the INM.Since non--functional INM targeting motif Trm1--II mutant contains an amino acid substitution, it raise a possibility that the non--functional INM targeting motif Trm1--II mutant may be a misfolded protein, and thereby affecting Trm1--II--lipid interactions.Further investigation are required
Author: Tsung‐Po Lai
Institution: The Ohio State University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2269075131