Interplay between two functions delivered by the N-terminal domain of MinE

1. The N-terminal domain of MinE contributes to both membrane association and stimulation of MinDATPase activity assembly: V+ > V- in PZ: V- ≥ VEr- - + 謝正偉 Cheng-Wei Hsieh, 林主祈 Chu-Chi Lin and 史有伶 Yu-Ling Shih Institute of Biological Chemistry, Academia Sinica, 128 Sec 2, Academia Road, Nankang, Taipei, 115, Taiwan [D]membrane VEr- V+ V- [D]cytosol Introduction Interplay between two functions delivered by the N-terminal domain of MinE During cell division, accurate positioning of the division plane ensures both progeny inherit equal amount of biological materials. Failure in this step can lead to serious consequences in all cell types. In E. coli, the cell division site determination is mediated by the Min system. The Min system is composed of three protein components (MinC, MinD, and MinE) that continuously oscillate between two ends of a cell to establish the appropriate site for cell division. MinD and MinE are sufficient of forming a dynamic oscillator in membrane-dependent and ATP-regulated manners. Our laboratory has demonstrated that the N-terminal domain of MinE can directly interact with the membrane through electrostatic interaction. However, the same region is also known to interact with MinD and further stimulate MinDATPase activity. In this study, we intend to examine the interplay between the two functions delivered by the N-terminal domain of MinE. (A) (B) (C) Asp45 Val49 • Figure 2. Illustration of the N- and C-terminal domains of MinE(A,B) and effects of charge mutations in the N-terminal domain on MinE’s ability to interact with MinD and stimulate of MinDATPase activity (C). The basic residues in MinE1-31 are highlighted in red. Although the C-terminal domain of MinE is know to form homodimers in protein structure (PDB entry 1EV0), the exact conformation of the N-terminal domain is uncertain. Residues Asp45 and Val49 are involved in formation of the MinE ring. Interactions between MinD and MinE were assayed by the bacterial two-hybrid system. The results in Miller units were converted to percentage of relative interaction by setting the wild-type interaction as 100%. 1 11 21 31 Malldfflsrkkntaniakerlqiivaerr r MinE1-31 Results 88 1 Co-localization of MinD and MinE defective in membrane association 32 MinE WT R10G/K11E/K12E R19G/K21E R29G/R30G/R31G Merge Aberrant oscillation cycles of MinD with the MinE charge mutants 1 32 (A) MinER10G/ K11E/ K12E 88 0’00’’ 10’00’’ 20’00’’ 26’00’’ 28’00’’ 31’00’’ 33’30’’ 36’29’’ 38’30’’ 39’29’’ 40’30’’ DIC MinEMinD 42’30’’ 44’44’’ 47’29’’ 50’30’’ 53’48’’ 57’33’’ 58’03’’ 58’48’’ 1’00’03’’ 1’04’49’’ Yfp-MinD (B) MinER29G/ R30G/ R31G 0’00’’ 0’10’’ 0’20’’ 0’30’’ 1’00’’ 1’10’’ 1’20’’ 1’40’’ 2’10’’ 2’20’’ 2’30’’ 2’50’’ 3’00’’ 3’10’’ 3’20’’ 3’40’’ 7’40’’ 8’50’’ 9’00’’ 9’10’’ 9’20’’ 9’30’’ 9’40’’ 10’00’’ 10’20’’ 10’40’’ MinE-Cfp • Figure 3. Effects of charge mutations in the N-terminal domain of MinE on oscillation cycles of MinD. The experiments were done by expressing Plac::yfp-minDminE-cfpin min cells. White arrows: MinD polar zones; cyan arrows: MinE ring. disassembly: VEr+> V+ [D]membrane [E]D [E]membrane MinE ring VEr+ V- VEr- Kinetic models of MinD zone assembly and disassembly V+ [E]cytosol [D]cytosol disassembly: [D]cytosol≈ 0, VEr-> V- (A) (B) - + DIC [D]membrane V- VEr- V+ [D]cytosol +: growing end of the MinD zone -: non-growing end of the MinD zone [D]cytosol: concentration of cytosolicMinD • [D]membrane: concentration of membrane-bound MinD • [E]cytosol : concentration of cytosolicMinE • [E]membrane: concentration of membrane-bound MinE • [E]D: concentration of MinE recruited to the membrane by MinD • Er-: without MinE ring • Er+: with MinE ring (C) • Figure 1. Effects of charge mutants in the N-terminal domain of MinE on the colocalization patterns of MinD and MinE by expressing Plac::yfp-minDminE-cfpin min cells. White arrows: MinD polar zones shorter than half cell length; yellow arrows: MinD polar zones longer than half cell length; cyan arrows: MinE ring. • Figure 4. Models describing kinetic properties of assembly and disassembly of the MinD zone. Conclusion Sequence conservation in the N-terminal domain of MinE homologues Fitting direct MinE-membrane interaction into molecular mechanism of the dynamic Min system Through this study, we confirmed that the N-terminal domain of MinE performs two functions in the Min system.The membrane associating property of MinE is delivered by three clusters of basic residues in its N-terminal domain. Mutations in R10/K11/K12 abolished membrane association but retained MinE’s ability to interact with MinD and stimulate Min D ATPase activity. This resulted in aberrant movement of MinD zones and unstable MinE rings. These phenotypes resembled the D45A/V49A mutant. Mutations in the most conserved K19 and R21 residues appeared to completely disrupt all functions of MinE. Mutations in R29/R30/R31 appeared normal in interaction with MinD but failed to stimulate MinDATPase activity. This resulted in slower oscillation of the MinD polar zones in cells. Evidence for their involvement in membrane association is illusive. Finally, the C-terminal domain of MinE functions in sequestering the N-terminal domain from associating with the membrane, thus providing regulatory function to the N-terminal domain. Summary of phenotypes on the charge mutants of MinE • Figure 5. Sequence Logo of the N-terminal domains of MinE from 128 bacterial species. Acknowledgement This work is supported by National Science Council (NSC96-2311-B-001-039) and Academia Sinica (AS97-FP-M02-2), Taiwan.