Biomedical Engineering Reference
In-Depth Information
Fig. 2
AP-1 recruited to peptidoliposomes forms high-molecular-weight complexes. (
a
) Peptidoliposomes
made of soybean lipids and presenting LY peptides were incubated with clathrin adaptors, Arf1, and GMP-
PNP. After fl oatation on a sucrose step gradient, the fl oated liposomes were solubilized with Triton X-100 and
centrifuged into a 10-25 % sucrose velocity gradient (
horizontal arrow
). Ten fractions were collected and
analyzed by immunoblotting for
-adaptin. For comparison, the nonfl oated fraction and the original adaptors
were analyzed in parallel. The positions of the sedimentation markers IgM (19S) and 40S ribosomes are indi-
cated. Individual AP-1 adaptors (
ʳ
300 kDa) have a sedimentation coeffi cient of 7.7S. AP-1 recruited to sorting
peptides shows oligomerization to high-molecular-weight complexes even in the absence of clathrin. (
b
) AP-1
recruitment from cytosol. Liposomes with or without LY peptides were incubated with cytosol, Arf1, and GMP-
PNP or GTP. After a fi rst step gradient fl oatation, the fl oated fraction 1 was solubilized with Triton X-100, sedi-
mented into a sucrose gradient, and analyzed for
∼
-adaptin as in panel
a
. Unlike AP-1 from purifi ed adaptors,
AP-1 from cytosol is recruited to liposomes with Arf1/GMP-PNP even in the absence of sorting peptides.
However, LY peptides are required for AP-1 to oligomerize. If GTP is used, stable recruitment is observed only
with sorting peptides. These results indicate the presence of cytosolic factor(s) recruiting AP-1 to membranes
in a cargo-independent, GTPase-sensitive manner. They further show that membrane recruitment is not suf-
fi cient to induce AP-1 oligomerization, but that binding to cargo signals is necessary. (Reprinted in modifi ed
form from ref.
10
with permission of The American Society for Cell Biology.)
ʳ
6. It is important not to contaminate the cytosol with the pellet.
Carefully remove the cytosol with a glass pipet, leaving behind
the last 1 cm of supernatant above the pellet.
7. To fi ll tubes to their minimal fi lling level or to balance them, a
1:1 mixture of buffers A and B may be added.
8. AP-2, AP180, and some clathrin are the major contaminants.
9. The later fractions of the Arf1 peak are usually purer than the
earlier ones.
10. The concentration of IPTG to induce maximal expression
and the optimal time and temperature of culturing for maxi-
mal yield are determined in small-scale cultures beforehand
by SDS-gel electrophoresis of lysed bacteria and Coomassie-
blue staining. For this, a 40-mL culture is grown to OD 0.7,
divided into four 10-mL cultures to be incubated with 0.5 or
1 mM IPTG at 30 °C or 37 °C. After 0, 1, 2, 3, and 4 h
2-mL samples were removed, pelleted, and lysed in 500
ʼ
L
loading buffer.
