Biology Reference
In-Depth Information
(0.2 M Na
2
CO
3
, pH 11) for extraction used for studying topologies of polytopic
membrane proteins is unsuitable as this method might denature proteins and/or strip
proteins from the LLD surface. We used hypotonic solution to swell and compromise
microsomal membrane integrity to release LLDs. Because LDs do not contain aque-
ous phase within the core, they are not susceptible to hypotonic osmotic pressure.
One of the challenges we have encountered in the preparation of LLDs is their
low abundance. Our studies have suggested that less than 3% of the intrahepatic
TG is contained in LLDs. This problem is compounded by the relatively low recov-
ery. For obtaining sufficient amount of LLDs for subsequent studies, usually two to
four mouse livers are necessary. However, it is challenging to adapt this method to
cultured hepatocytes unless one uses radioactive tracers to monitor LLD recovery.
As mentioned above, an alternative approach to release microsomal lumenal con-
tents is through the use of high pH Na
2
CO
3
(
Sundaram et al., 2010
). This method
is more efficient at breaking microsomes than the hypotonic method we used, and
therefore results in a higher recovery of lumenal contents. However, because
Na
2
CO
3
will also strip proteins and CLDs peripherally associated with membranes
in addition to LLDs, this might result in contamination of LLDs with CLDs that
remained associated with microsomes. Readers should choose carefully which
procedure is preferable depending on the experimental needs. The following
publications maybe referred to for comparison (
Wang et al., 2007; Yao, Zhou,
Figeys, Wang, & Sundaram, 2013
).
To assess the purity of the isolated LLDs and to estimate the degree of contam-
ination, protein composition needs to be determined. The LLD fractions should
be free of apoB, transmembrane proteins, and CLD markers. However, many ER
lumenal proteins are found in LLDs. This may be due to the ER-LD connection,
as by high-confidence LD proteomics studies, many proteins were found to have dual
localization in the ER and on LDs (
Krahmer et al., 2013
). Using mass spectrometry,
we also found some cytosolic and mitochondria associated proteins in LLDs fraction.
These are inevitable contaminations that exist in essentially all subcellular purifica-
tions. Thus, further combining LLD purification with a high-confidence proteomics
approach such as SILAC would be beneficial to identify bona fide LLD proteins.
This approach would further assist in identifying specific protein markers for LLDs.
So far, proteins confirmed to be present on mouse liver LLDs are carboxylesterases 1
and 3, apoE, MTP (
Wang et al., 2007
), and apoCIII (
Sundaram et al., 2010
). How-
ever, these proteins are not exclusively associated with LLDs but are also present in
their “lipid-free” form in the ER lumen.
The isolation and analysis of LLDs provided direct biochemical evidence that
LLDs are true entities that possess different biochemical properties from that of
CLDs and VLDL. This protocol can be adapted to a wide variety of applications
to study the mechanism of LLD formation, the role of LLD-associated proteins in
VLDL secretion, as well as genetic, nutritional, and pharmaceutical influence on
these processes.
This protocol also provides an example of using BODIPY fatty acid analogues to
study dynamics of LD formation and metabolism. BODIPY fatty acids are available
