Biology Reference
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bodies. These internal vesicles contain receptor-ligand complexes that
are destined for lysosomal degradation.
72
Material en route to lyso-
somes (pH 4.5-4.0) is exposed to an increasingly acidic pH during
transport from early endosomes (pH 6.5-6.0) to EVC and late endo-
somes (pH
≤
5.6).
66,68,73,74
Budding of ECV from early endosomes
requires their acidification and the presence of distinct coat proteins.
71
Thus, inhibition of the V-ATPase by bafilomycin selectively blocks
transport of cargo destined for lysosomal degradation in early endo-
somes. ECV are subsequently transported along microtubules to late
endosomes. Disruption of microtubules by nocodazole results in
accumulation of endocytosed material in ECV.
75
Finally, incubation of
the cells at 20
C results in retention of cargo in late endosomes and
thus prevents degradation in lysosomes.
76
Drugs and temperature can
thus be used to arrest internalized material in different compartments
on their way to lysosomes depending on the cell type (Fig. 4). These
treatments also have distinct effects on the recycling pathway. This
route is taken by (iron-loaded) transferrin and the transferrin recep-
tor.
77
Therefore, the transferrin pathway is most often used for con-
trol purposes in investigations of virus entry pathways.
The transferrin-transferrin receptor complex is taken up via clathrin-
coated pits and vesicles into early endosomes, where the iron is
released.
77
The resulting apo-transferrin remains bound to the receptor
and is recycled and subsequently released into the neutral extracellular
milieu. Transferrin recycling can occur via two pathways: from early
endosomes with t
1/2
≈
°
2.5 min (fast) and from the perinuclear recycling
compartment (PNRC) t
1/2
≈
7 min (slow).
78,79
The pH of the PNRC is
higher than that of early endosomes in CHO and Hep2 cells, whereas
the PNRC is more acidic than early endosomes in HeLa cells.
73,77
Biochemically, the PNRC is distinct from early endosomes as well as from
ECV and late endosomes. It contains recycling receptors such as LDLR,
but no material that is targeted to lysosomes.
77
We have recently shown
that the kinetics of transferrin acidification in HeLa cells is biphasic,
indicative of fast and slow recycling pathways via early endosomes
(pH 6.0) and the PNRC (pH 5.6). Nocodazole blocks the second phase
of transferrin acidification, demonstrating that transferrin transport from
