254
6. The psd module constructs are expressed by a constitutive
promoter, which leads to constant synthesis of the psd module. Under blue light illumination, a steady-state level is
reached, which depends on production and degradation rates
of the protein. Usage of a regulatable promoter may further
decrease the depletion time and lower target protein abundance at restrictive conditions.
7. A suitable control to assess the influence of a certain mutation
on the degradation mechanism of the construct under consideration is a mutation that abolishes cODC1 degron activity.
For constructs with differences in light-regulated activation of
the degron, a similar stability is expected after inactivation of
the degron. If a mutation in the photoreceptor domain influences the stability of the construct without increasing the
degron presentation, the increased degradation rate persists
after mutational degron inactivation. Such a mutation is less
valuable, as the contribution of the mutation is not controlled
by light and cannot be influenced by further modifications of
the photoreceptor. Ideally, all modifications influence the degradation rate solely by impacting on degron presentation. In
this case, the signaling state of the photoreceptor determines
protein stability.
8. Another set of controls are mutations that abolish lightdependent signaling of the photoreceptor. For the A. thaliana
phototropin 1 LOV2 domain examples are I608E and C512A,
which lock the photoreceptor in the light and the dark state,
respectively. Please note that for mutations similar to C512A
some residual light activation has been observed in homologous LOV domains [31].
Acknowledgments
I thank D Störmer for her excellent technical assistance as well as S
Hepp, C Renicke, and J Trauth for helpful comments on the manuscript. A Batschauer is acknowledged for the opportunity to use
his photobiology equipment. This work was supported by the
DFG grant TA320/3-1 and the DFG-funded graduate school
GRK1216.
References
1. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479
2. Pickart CM (2001) Ubiquitin enters the new
millennium. Mol Cell 8(3):499–504
3. Ravid T, Hochstrasser M (2008) Diversity of
degradation signals in the ubiquitin- proteasome
system. Nat Rev Mol Cell Biol 9(9):679–690
4. Dohmen RJ, Wu P, Varshavsky A (1994) Heatinducible degron: a method for constructing
temperature-sensitive
mutants.
Science
263(5151):1273–1276
5. Banaszynski LA, Chen LC, Maynard-Smith
LA, Ooi AG, Wandless TJ (2006) A rapid,
reversible, and tunable method to regulate
Christof Taxis
6. The psd module constructs are expressed by a constitutive
promoter, which leads to constant synthesis of the psd module. Under blue light illumination, a steady-state level is
reached, which depends on production and degradation rates
of the protein. Usage of a regulatable promoter may further
decrease the depletion time and lower target protein abundance at restrictive conditions.
7. A suitable control to assess the influence of a certain mutation
on the degradation mechanism of the construct under consideration is a mutation that abolishes cODC1 degron activity.
For constructs with differences in light-regulated activation of
the degron, a similar stability is expected after inactivation of
the degron. If a mutation in the photoreceptor domain influences the stability of the construct without increasing the
degron presentation, the increased degradation rate persists
after mutational degron inactivation. Such a mutation is less
valuable, as the contribution of the mutation is not controlled
by light and cannot be influenced by further modifications of
the photoreceptor. Ideally, all modifications influence the degradation rate solely by impacting on degron presentation. In
this case, the signaling state of the photoreceptor determines
protein stability.
8. Another set of controls are mutations that abolish lightdependent signaling of the photoreceptor. For the A. thaliana
phototropin 1 LOV2 domain examples are I608E and C512A,
which lock the photoreceptor in the light and the dark state,
respectively. Please note that for mutations similar to C512A
some residual light activation has been observed in homologous LOV domains [31].
Acknowledgments
I thank D Störmer for her excellent technical assistance as well as S
Hepp, C Renicke, and J Trauth for helpful comments on the manuscript. A Batschauer is acknowledged for the opportunity to use
his photobiology equipment. This work was supported by the
DFG grant TA320/3-1 and the DFG-funded graduate school
GRK1216.
References
1. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479
2. Pickart CM (2001) Ubiquitin enters the new
millennium. Mol Cell 8(3):499–504
3. Ravid T, Hochstrasser M (2008) Diversity of
degradation signals in the ubiquitin- proteasome
system. Nat Rev Mol Cell Biol 9(9):679–690
4. Dohmen RJ, Wu P, Varshavsky A (1994) Heatinducible degron: a method for constructing
temperature-sensitive
mutants.
Science
263(5151):1273–1276
5. Banaszynski LA, Chen LC, Maynard-Smith
LA, Ooi AG, Wandless TJ (2006) A rapid,
reversible, and tunable method to regulate
Christof Taxis