Biology Reference
In-Depth Information
Microdomains
In addition to the plasma membrane, detergent-insoluble microdomains have
been identified in other cellular membranes including mitochondrial memb-
ranes (
Christie et al., 2012; Ciarlo et al., 2010; Sorice et al., 2009
). In the mitochon-
dria, cardiolipin is the major phospholipid and evidence suggests that there exist
microdomains selectively enriched in cardiolipin (
Osman, Voelker, & Langer,
2011
). Cardiolipin is a phospholipid that has been shown to be important for
optimal function of the mitochondrial respiratory chain. Thus, it is possible that
the respiratory chain operates within cardiolipin-enriched microdomains in the
inner mitochondrial membrane. Isolation of these microdomains will be impor-
tant for the biochemical characterization of respiratory chain complexes and super-
complexes, and to determine their biological relevance.
In this chapter, we will concentrate on the characterization of multimolecular
complexes and supercomplexes in detergent-insoluble microdomains from mito-
chondrial membranes, specifically cardiolipin-enriched microdomains. These mo-
lecular structures are a focus of intense study, specifically understanding how
they are assembled and how they ensure optimal cellular respiration. There is an
increasing amount of experimental evidence suggesting that mitochondrial
respiratory complexes are not simply randomly dispersed within the inner mito-
chondrial membrane but rather undergo ordered supramolecular associations. These
mitochondrial respiratory supercomplexes were first identified by Schagger and
von Jagow when they performed blue-native gel electrophoresis on digitonin-
solubilized yeast and mammalian mitochondria (
Schagger & Pfeiffer, 2000;
Schagger & von Jagow, 1991
). Since then, supercomplexes have been found in
many mammalian tissues, as well as other organisms including fish, fungi, and
bacteria (
Lenaz & Genova, 2012
). These supercomplexes mainly consist of the
standard respiratory complexes I, III, and IV in various stoichiometries (e.g.,
I-III
2
-IV
1-4
), though there may be some cases in which complex II is involved
(
Acin-Perez, Fernandez-Silva, Peleato, Perez-Martos, & Enriquez, 2008
). Arrange-
ment of respiratory complexes into supercomplexes has been suggested to aid in
substrate channeling and may be important for optimal activity of the respiratory
chain. Furthermore, there is evidence that cardiolipin may be necessary for super-
complex assembly (
Pfeiffer et al., 2003
). The reader is referred to several excellent
reviews on mitochondrial respiratory supercomplexes for further details (
Lenaz &
Genova, 2009, 2012
).
Here, we will describe methodology for isolating various membrane micro-
domains, including detergent-insoluble glycosphingolipid microdomains from
plasma membranes for biochemical characterization, and methods for
isolating cardiolipin-enriched mitochondrial membranes and native protein com-
plexes including the mitochondrial respiratory chain supercomplexes. These
techniques will provide the tools necessary to effectively study multimolecular
complexes and oligomeric structures in their intrinsic membrane location and
explore the biological functions associated with these specialized membrane
microdomains.
