Biology Reference
In-Depth Information
2006; Kohlwein et al., 2013
), or mitochondria (
Pu et al., 2011
), as well as the iden-
tification of novel factors influencing biogenesis and dynamics of LD (
Adeyo et al.,
2011
) accentuates LD as a central topic in cellular biology.
Although occurrence and structure of LD are similar in all eukaryotes, there
are some differences in the lipid composition and the set of proteins, even in
different yeast species and in strains grown on different carbon sources. Mass
spectrometric analysis of lipids and proteins of LD from
S. cerevisiae
cultivated
on glucose and oleate, respectively, revealed that LD proteome and lipidome can
adapt to environmental changes (
Grillitsch et al., 2011
). When cultivated on
oleate, peroxisomes proliferate which are the only organelle of the yeast where
b
-oxidation of FA occurs. Under these growth conditions, accumulation of nonpolar
lipids was observed accompanied by an altered ratio of TG to SE. Oleate
stimulates the formation of TG at the expense of SE in
S. cerevisiae
which is in sharp
contrast to
Yarrowia lipolytica
(
Connerth et al., 2010; Rosenberger, Connerth,
Zellnig, & Daum, 2009
). This effect is only one example for differences
observed with LD from the yeasts
S. cerevisiae
,
Pichia pastoris
, and
Y. lipolytica
.
LD of the oleaginous yeast
Y. lipolytica
vary in size from 650 to 2500 nm
depending on cultivation conditions and are markedly larger than LD from
S. cere-
visiae
(
Athenstaedt et al., 2006
). It was also shown that not only size and
abundance of LD from
Y. lipolytica
depend on the carbon source but also the lipid
composition and the proteome. Further examples for such effects will be described in
Section 3
.
To obtain highly pure LD from
S. cerevisiae
,
P. pastoris
, and
Y. lipolytica
, iso-
lation protocols were established as will be described in
Sections 3 and 4
. Besides the
protocol for the isolation of LD at high purity we will present quality control by
Western blot analysis adapted to different requirements of the different yeasts. Fur-
thermore, we will discuss the analysis of proteins and lipids from LD based on thin
layer chromatography (TLC), gas liquid chromatography (GLC), mass spectrometry
(MS) as well as GLC/MS. Finally, we will briefly compare LD proteome and lipi-
dome from the three different yeasts
S. cerevisiae
,
Y. lipolytica
, and
P. pastoris
(
Athenstaedt et al., 2006; Grillitsch et al., 2011; Ivashov et al., 2012
).
2.1
MATERIALS
2.1.1
Equipment and supplies
Incubator (Heraeus, Thermo Fisher Scientific Inc., Waltham, Massachusetts,
USA)
Table top centrifuge (HettichRotina 46R, Heraeus Fresco 17)
Sorvall RC6 plus centrifuge (Thermo Fisher Scientific Inc., Waltham,
Massachusetts, USA)
Sorvall Ultracentrifuge Combi (Thermo Fisher Scientific Inc., Waltham,
Massachusetts, USA)
