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2.3.2 Proteome analysis of LD
Proteome analysis of LD from S. cerevisiae , P. pastoris , and Y. lipolytica revealed
that LD proteins can be classified into certain functional families ( Athenstaedt et al.,
2006; Fei, Zhong, et al., 2011; Grillitsch et al., 2011; Ivashov et al., 2012 ). Enzymes
of lipid metabolism comprise the biggest group next to glycosylation and protein
synthesis, cell wall organization, and ER-unfolded protein response. The most
abundant LD proteins from S. cerevisiae are Ayr1p, Dga1p, Eht1p, Erg1p, Erg27p,
Erg6p, Erg7p, Faa1p, Faa4p, Fat1p, Gat1p, Hfd1p, Pet10p, Pgc1p, Slc1p, Tgl1p,
Tgl3p, Tgl4p, Tgl5p, Tsc10p, and Vps66p. The number of LD proteins detected
in P. pastoris ( Ivashov et al., 2012 ) and Y. lipolytica ( Athenstaedt et al., 2006 ) iden-
tified so far is low compared to S. cerevisiae . Different abundance of proteins in dif-
ferent yeast genera but also different methods employed for proteome analysis
may be the reason for this observation. For an overview of proteome analysis of
the different yeasts the reader is referred to the abovementioned publications.
The proteome of S. cerevisiae shows an adaptive response to environmental con-
ditions. As an example, additional LD proteins have been found in cells grown on
oleate compared to growth on glucose ( Grillitsch et al., 2011 ). Fei, Zhong, et al.
(2011) reported that the LD proteome is influenced by size and phospholipid com-
position of the droplets as shown with yeast mutants producing “supersized” LD.
Differences in the LD proteome between the investigated yeast species and caused
by variation of cultivation conditions led to the speculation that a basal set of LD
proteins is sufficient to maintain structure and function of this organelle.
Structural and topological investigations of LD proteins as well as targeting of
proteins to this organelle are just in their infancy ( Hickenbottom, Kimmel,
Londos, & Hurley, 2004 ). Initial experiments to address this issue led to the conclu-
sion that hydrophobic domains near the C-terminal end of LD proteins may play a
role in their distribution between LD and the ER as demonstrated for Erg1p, Erg6p,
and Erg7p ( M¨ llner, Zweytick, Leber, Turnowsky, & Daum, 2004 ). Another inter-
esting feature of LD proteins seems to be that they do not harbor transmembrane
spanning domains. This property can be explained by the fact that LD proteins need
to be accommodated in the surface phospholipid monolayer of the organelle.
2.3.3 Lipid analysis of LD
Nonpolar lipids of LD can be routinely analyzed by TLC and identified by compar-
ison to standard mixtures. Figure 2.2 shows a typical analysis of nonpolar lipids from
LD samples and standards as mentioned in Section 2 . For the TLC shown in Fig. 2.2 A
lipids were separated by a two-step procedure using light petroleum/diethyl ether/
acetic acid (35:15:1; per vol.) as a first solvent system and light petroleum/diethyl
ether (49:1; v/v) as a second solvent system. Two micrograms of cholesteryl oleate,
triolein, and ergosterol, respectively, and 0.15 m g protein equivalent of LD sample
from S. cerevisiae were loaded. As shown in Fig. 2.2 A, LD from S. cerevisiae contain
approximately equal amounts of TG and SE. It is worth mentioning that SE and TG
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