Biology Reference
In-Depth Information
FIGURE 2.1
Quality control of lipid droplets (LD) isolated from S. cerevisiae. Cells were grown to the
stationary phase on glucose medium. Western blot analysis of 10
g protein from each
fraction was performed as described in
Section 2
. Homogenate (H), LD (LD), 30,000
m
g
microsomes (M30), 40,000
g microsomes (M40), mitochondria (M), and cytosol (C).
Antibodies were directed against marker proteins from S. cerevisiae. Erg6p (sterol
24-C-methyltransferase; LD marker), Wbp1p (dolichyl-diphosphooligosaccharide-protein
glycotransferase; ER marker), Cyb2p (cytochrome b
2
;
L
-lactate cytochrome-c
oxidoreductase; mitochondrial marker), and GAPDH (glyceraldehyde-3-phosphate
dehydrogenase; cytosolic marker).
analysis using a typical set of antibodies directed against marker proteins from
S. cerevisiae
is shown in
Fig. 2.1
. Antibodies against GAPDH and Erg6p can also
be used for the quality control of subcellular fractions isolated from
P. pastoris
and
Y. lipolytica
. Alternatively to Cyb2p, an antibody against the porin Por1p can
be used as marker for the mitochondria fraction. Additionally, marker proteins fused
to a Myc-, HA-, or a GFP (green fluorescent protein)-tag can be used to check the
purity of different fractions if suitable antibodies are not available.
However, due to the tight interaction and contacts of LD to other organelles like
the ER, mitochondria, and peroxisomes (
Kohlwein et al., 2013
) on one hand, and
slight contamination of LD with other subcellular fractions which can never be
avoided on the other hand, highly sensitive MS proteome analyses do not only detect
“true” LD proteins, but also contaminations.
Krahmer et al. (2013)
recently reported
a novel methodology for LD protein identification based on MS and the so-called
protein correlation profiling. This profile allows the identification of LD proteins
with high confidence by using quantitative, high-resolution MS and by correlating
their purification profile to that of known LD proteins.
In addition to MS analysis, the presence of many proteins in LD was confirmed
by fluorescence microscopy studies. SDS-PAGE revealed different protein patterns
of LD fractions depending on the cultivation conditions, for example when glucose
or oleate was used as carbon source. This effect was observed with
S. cerevisiae
and
Y. lipolytica
(
Athenstaedt et al., 2006; Grillitsch et al., 2011
).
