M2 Structural and Functional Biochemistry (SFB) – Internship proposal 2018
Characterisation of TIM23 complex (Translocase of the mitochondrial Inner membrane)
to tackle protein sorting in mitochondria
Supervisors:
Anne
SARCOS
([email protected])
&
Raffaële
IEVA
([email protected])
Laboratory: Group “Regulation and transport of proteins across cell membranes”
Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), UMR CNRS/UPS 5100, Bâtiment IBCG,
118, route de Narbonne, 31 077 Toulouse
Mitochondria are essential organelles of eukaryotic cells. Most mitochondrial proteins are encoded by
nuclear genes and synthesized in the cytosol as preproteins with an amino-terminal mitochondriatargeting presequence. The presequence translocase of the mitochondrial inner membrane (TIM23
complex) imports preproteins either into the matrix or into the inner membrane. The initial transport
of presequence signals by TIM23 is energized thanks to the electrochemical potential established across
the inner membrane by the electron transport chain complexes. To import soluble preproteins into the
matrix, TIM23 dynamically interacts with the ATP-driven presequence translocase associated motor
(PAM). On the other hand, preproteins with a hydrophobic inner membrane-targeting segment are
laterally released into the lipid phase in a step that does not require PAM. This M2 internship project
focuses on the mechanisms by which TIM23 recognizes the presequence and other signals of
preproteins to ensure their correct sorting. The essential protein Tim50 acts as a presequence binding
receptor of TIM23 and leads to the opening of the translocating channel. It is unclear, however, whether
Tim50 plays a role in directing precursors to the correct compartment after presequence recognition.
Recent work in the lab reveals that Tim50 is required for import steps that occur subsequently to the
initial presequence binding.
Budding yeast will be used as a model system to study how Tim50 variants (encoded by temperature
sensitive alleles) affect the organization of TIM23 with PAM to promote import into the matrix. Methods
such as native immune-precipitations, blue-native PAGE, site-directed photocrosslinking will be used to
monitor and map TIM23-PAM interactions at sequential steps of the import reaction. A structurefunction map of Tim50 will be built combining biophysical analysis of TIM23-PAM interactions,
functional import assays, and structural modeling of Tim50 mutant variants. This work will help to
understand the molecular mechanisms that ensure correct protein sorting into mitochondria.
Methods:
cloning,
mutagenesis,
affinity
chromatography,
blue-native
PAGE,
site-directed
photocrosslinking, biophysics analyses of protein complexes
References
 Schendzielorz AB, Schulz C, Lytovchenko O, Clancy A, Guiard B, Ieva R, van der Laan M, Rehling P. (2017)
Two distinct membrane potential-dependent steps drive mitochondrial matrix protein translocation.
J Cell Biol. 216:83-92.
 Turakhiya U, von der Malsburg K, Gold V, Guiard B, Chacinska A, van der Laan M, Ieva R*. (2016) Protein
import by the mitochondrial presequence translocase in the absence of a membrane potential. J Mol
Biol, 428:1041-52.
 Ieva R, Schrempp SG, Opaliński L, Wollweber F, Höss F, Heißwolf AK, Gebert M, Zhang Y, Guiard B,
Rospert S, Becker T, Chacinska A, Pfanner N, and van der Laan M. (2014) Mgr2 functions as lateral
gatekeeper for preprotein sorting in the mitochondrial inner membrane. Mol Cell, 56:641-52.