were influenced by ONO treatment. In HeLa cells stably expressing
an LDLR-GFP construct and incubated with LDL-DiI for 5 min at
37 C, both LDLR and LDL co-localized in puncta representing
peripheral SEs (Fig. 4A and B). As expected, under these conditions
of 5 min uptake, LDLRs were also found in later endocytic compartments
that did not contain LDL-DiI. When HeLa-LDLR-GFP cells
were pulsed with LDL-DiI for 5 min followed by a 40 min chase
in 10 lM ONO, both LDLR and LDL remained localized to these
peripheral SEs (Fig. 4C and D). However, when these cells were
pulsed with LDL-DiI for 5 min followed by a 40 min chase in
1 lM ONO, LDLR and LDL localized in the juxtanuclear region,
but they were found in different populations of vesicles (Fig. 4E
and F). Additional evidence that LDLR and LDL-DiI are in different
compartments was obtained by showing that BFA treatment
caused the LDLR-positive compartments to form tubules, but LDL
was still localized in puncta throughout the cytoplasm (Fig. 4G
and H). These results show that ONO also caused a concentration-
dependent block in the normal trafficking of both LDLR and
LDL: first at high concentrations at a common step in peripheral
SEs; and, second, at low concentrations in separate compartments
that are downstream from SEs. Similar results were obtained with
the PLA2 antagonist BEL (data not shown).