Download Antiporter-lika proteinsubenheter i andningskedjans Komplex I

Examensarbete i kemi, naturvetenskapliga fakulteten, Lunds universitet
Antiporter-lika proteinsubenheter i andningskedjans Komplex I
Benoit Combaluzier
All organisms need energy to survive. The energy comes either from sunlight
(photosynthesis) or from burning food molecules (respiration), but in both cases the energy is
converted to ATP by the organism. ATP is an energy stock that can be used by a multitude of
processes in the cell. Eukaryote aerobic organisms produce ATP in the mitochondria by an
assembly of protein complexes called respiratory chain or electron transport chain. The
respiratory chain is composed by 4 protein complexes which pump protons across the
membrane and a fifth complex that uses this proton gradient to build ATP from ADP and Pi.
The Complex I, also called NADH:quinone oxidoreductase, is the least understood in the
respiratory chain. The bacterial Complex I is composed by 14 protein subunits. The
mammalian Complex I has 32 accessory subunits. Since the bacterial Complex I is a simple
model, it has been chosen to study the structure and the function of Complex I. It is not
understood how the complex works on the molecular level and how electron transfer through
Complex I drives proton pumping across the membrane.
Three subunits, NuoL, NuoM and NuoN, located in the membrane part of the complex I have
structure similarities to one class of antiporters. Antiporters are membrane proteins in charge
of transporting ions across the membranes. These antiporters, called MrpA and MrpD, are
responsible for salt balance and for maintaining a neutral cytoplasmic pH at alkaline growth
conditions.
Since NuoL, NuoM and NuoN show similarities to that kind of antiporters, they are most
likely involved in the proton transport machinery. NuoL is more similar to MrpA and NuoM
and NuoN are more similar to MrpD.
Bacillus subtilis lacking the MrpA protein cannot grow at high Na+ concentration but they
could be rescued if NuoL protein was expressed in vivo. The same observations were made
with MrpD-deleted strains rescued by NuoM. This demonstrated that NuoL and NuoM have
Na+ transport ability.
In this work, we cloned the NuoN, MrpA and MrpD genes into a plasmid DNA vector. The
plasmid containing the gene encoding for NuoN protein could be used to investigate if NuoN
has also an ion transport ability and can replace MrpD in B. subtilis lacking MrpD protein.
The plasmids containing MrpA and MrpD genes could be used to control that the cells lacking
MrpA or MrpD in their genome can retrieve their functionality when these proteins are
expressed from the plasmids.
Swedish official title: Antiporter-lika proteinsubenheter i andningskedjans Komplex I
Swedish credits: 20p
E-mail address of first author: [email protected]
Supervisor: Cecilia Hägerhäll, Biochemistry
Submission date/time: 5/4/2004
Examensarbete i kemi, naturvetenskapliga fakulteten, Lunds universitet
Antiporter-like subunits in respiratory chain complex I
Benoit Combaluzier
Chemistry, Biochemistry
Autumn 2003
Abstract in English
NADH:quinone oxidoreductase or Complex I is the largest and most complex of the
respiratory chain enzymes. The enzyme oxidizes NADH to NAD+ and reduces quinone to
quinol. Electron transfer through the enzyme is coupled to proton translocation by an
unknown mechanism. The membrane spanning part of the enzyme contains three subunits,
NuoL, NuoM and NuoN that show primary sequence similarity to a certain type of
antiporters, MrpA and MrpD, and are thus predicted to play a role in the proton translocation
machinery. NuoL is more closely related to MrpA and NuoM and NuoN are more closely
related to the MrpD antiporter. In a previous work the function of the NuoL and NuoM
subunits from Rhodobacter capsulatus Complex I has been investigated by expressing the
proteins in Bacillus subtilis strains where the homologous MrpA and MrpD antiporters had
been genetically deleted. It appeared that NuoL and NuoM proteins have Na+ translocation
ability in vivo.
In this work we will clone the MrpA, MrpD and NuoN genes and express the proteins in the
two deletion strains. We could then compare the growth of Bacillus subtilis MrpD-deleted
strain alone or with expression of NuoN from the pCW6 vector under several pH conditions.
We could also check that the functionality is recovered by Bacillus subtilis MrpD-deleted
strain when MrpD is expressed from the pCW6 vector. The same study could be carried out
with Bacillus subtilis MrpA-deleted strain.