requires concerted action of AXIN, the lysosomal v-ATPase, and
the Ragulator complex (a pentamer consisting of LAMTORs 1-5)
[15, 17]. The scaffold protein AXIN tethers LKB1 by virtue of its
ability to directly interact with the upstream kinase, to AMPK
located on the surface of lysosome. The lysosomal v-ATPase-Ragu-
lator complex plays an important part in the docking of AXIN/
LKB1, allowing for the formation of the stable complex of
v-ATPase-Ragulator-AXIN/LKB1-AMPK on the surface of lyso-
some [18] and the ultimate activation of AMPK by LKB1. More-
over, subcellular fractionation identified that the AMPK-activating
complex resides on the lysosomal detergent-resistant membrane
(DRM) which is known as lipid raft [15, 19].
To obtain genetic evidence whether a given AMPK activation is
through the lysosomal pathway,AXIN/and LAMTOR1/
mouse embryonic fibroblasts (MEFs) or mouse individuals are
starved of glucose or food or treated with other metabolic stresses
and pharmacological stimuli. The various cells or animal tissues can
be analyzed for AMPK activity by assessing p-Thr-172-AMPKαand
p-Ser-79-ACC. As mentioned above, the lysosome is where AMPK
phosphorylation occurs after AXIN in complex with LKB1 interacts
with v-ATPase-Ragulator. Therefore, isolation of lysosomes of dif-
ferent purity is a direct and efficient way to monitor the “turning
on” and “turning off” of the lysosomal pathway (Fig.1). In addi-
tion, it has been well established that Ragulator complex is localized
on the DRM region (lipid raft) of lysosome [19–21]. In this proto-
col, we describe how to isolate intact lysosomes and their DRMs by
using the method originally developed by Sigma-Aldrich LLC and
Kawabuchi et al. [22]. Through these methods, the dynamic asso-
ciation of the AXIN/LKB1 with the lysosomal DRM, as well as the
phosphorylation status of AMPK, in animal tissues or cells can be
conveniently determined by immunoblotting after immunoprecip-
itation (IP) [15]. We also describe the procedures for immunofluo-
rescent staining to visualize the complex formation in intact cells.
Although the above-described method for isolating lysosome
and DRM has allowed us to analyze specific protein components on
the membrane before and after glucose starvation, they are “less
active” and are not suitable for reconstituting the binding of
AXIN/LKB1 to the lysosome in vitro. To obtain active membrane
structures, Steinberg et al. [23] and Zoncu et al. [24] brilliantly
developed a method to isolate crude cytosolic membranes, named
as light organelles, in a relatively mild condition. The light orga-
nelles have been applied to investigate mechanisms for regulation of
mTORC1, which is also activated through v-ATPase and Ragulator
[20, 24]. We have demonstrated that the light organelles isolated
from glucose-starved cells and cells cultured in complete medium
preserve activities for a series of in vitro reconstitution assays for
AMPK regulation through the lysosomal pathway [15].394 Chen-Song Zhang et al.