Sorting isozymes are encoded by
single genes, but are distributed to multiple subcellular
compartments. We compared genes for 5 eukaryotic sorting isozymes
with the archaeal/eubacterial counterparts and found that eukaryotic
genes possess extra sequences. Those additions that have been studied
serve subcellular targeting roles and, thus, we term the additions
'ADEPTs' -additional domains for eukaryotic protein targeting
(Stanford et al. 2000). Trm1p is a sorting isozyme. Nuclear Trm1p is
peripherally associated with the inner nuclear membrane (INM; Rose et
al. 1995). We employed the ADEPT concept to predict the Trm1p motif
responsible for its INM location. Trm1p contains 4 'extra'
sequences. The N-terminal addition is a mitochondrial targeting
sequence; addition 2 (aa 91-136) includes the NLS. Deletion of the 3rd
addition (aa 346-367) causes Trm1-GFP to be nucleoplasmic. Thus, ADEPT
3 appears to be necessary for INM location. Addition of sequences
including ADEPT 3 to a nucleoplasmic reporter causes the reporter to
locate at the INM. So, ADEPT 3 appears to be sufficient for INM
location. We are mapping the aa that specify INM location and we are
searching for trans-acting mutations that mislocate Trm1-GFP. So far,
we identified mutants in which Trm1p is located at an ER-like
structure or at 1-2 nuclear membrane spots. We are testing the
hypothesis that the mutants define an ER to INM pathway for nuclear
proteins. (Funded by NIH and NSF grants to AKH and NCM).
Return to YGM 2000 Abstract Index