(Received for publication, January 24, 1995)
From the
It is well established that a proportion of newly synthesized lysosomal enzymes and class II major histocompatibility complex antigens are delivered directly to the endocytic pathway from the Golgi complex. Here we show that a significant proportion of newly synthesized transferrin receptors can be detected in endosomes before reaching the cell surface. These newly synthesized transferrin receptors are delivered to the endosome more efficiently than either constitutively secreted soluble proteins or glycophosphatidylinositol-anchored plasma membrane proteins suggesting that their transfer to the endosome is signal-dependent. Identification of a signal-dependent transfer step for proteins like the transferrin receptor operating on the exocytic pathway has important implications for membrane biogenesis, especially in the establishment of cell surface polarity.
Newly synthesized proteins trafficking to the cell surface pass
through the Golgi stack en route to a variety of destinations on the
cell surface and inside the cell. Intracellular destinations include
locations along regulated secretory pathways and endocytic routes. The
earliest decisions in this selective routing are taken in trans-Golgi elements(1) , many of which contain
clathrin coated domains whose ability to concentrate and selectively
route subsets of trafficking proteins is well documented(2) .
As yet, however, clathrin-coated domains are the only sorting
mechanisms which have been definitively shown to be capable of
selectively routing trafficking proteins in the TGN, ()and
in view of the variety of alternative routes known to originate in this
compartment it has become important to define more precisely the basis
on which this selection is made. It seems likely, for example, that the
first step in the sorting process is primarily concerned with selecting
proteins carrying signals recognized by clathrin-coated domains so that
further selective decisions are left to be made downstream, in the
recycling pathways which receive these proteins(3) .
The best documented routes running directly from the trans-Golgi to intracellular destinations are those which carry mannose 6-phosphate receptors and major histocompatibility class II antigen-invariant chain complexes to the endocytic pathway(4) . These proteins are selected in the clathrin-coated domains of the trans-Golgi, and current evidence suggests that their recognition depends upon signals being carried in their cytoplasmic domains(4) . Here we determine if other newly synthesized membrane proteins which carry recognition signals for clathrin lattices in their cytoplasmic domains are also delivered to endosomal compartments directly from the Golgi.
To
determine the rate at which newly synthesized Tfn-R and alkaline
phosphatase are transferred from the Golgi to the cell surface cells
were pulsed with trans-S-label (150 µCi/ml)
for 15 min in methionine-free medium and then chased in DMEM containing
10% FCS and 20 mM HEPES at 20 °C for 4 h. Cells were then
transferred to 37 °C for various times before cooling to 4 °C.
To detect Tfn-R at the cell surface, cells were then incubated in 100
µg/ml trypsin for 30 min at 4 °C(11) . The cleaved
fragment of the extracellular domain of the Tfn-R and the intact
receptor that remained cell associated were immunoprecipitated with
Rb9. Immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis followed by quantitation either by densitometry or
using a PhosphorImager (Bio-Rad, UK).
To detect alkaline phosphatase at the cell surface, cells were incubated with 0.5 units/ml phosphatidylinositol-specific phospholipase C for 2 h at 4 °C. Alkaline phosphatase released and that remaining cell-associated was immunoprecipitated with anti-alkaline phosphatase antibody and analyzed as described for the Tfn-R.
Horseradish peroxidase
activity in the endosome fraction and in an aliquot of the post-nuclear
supernatant was measured after solubilization in 1% Triton X-100 and
centrifugation at 100,000 g for 1 h (to remove gold
conjugates because they interfered with the horseradish peroxidase
assay).
To determine whether newly synthesized Tfn-R and alkaline
phosphatase pass through endosomes en route to the cell surface, cells
were pulsed with trans-S-label (150 µCi/ml)
for 15 min in methionine-free medium and then chased in DMEM containing
10% FCS and 20 mM HEPES at 20 °C for 4 h.
Anti-Tfn-R
gold was added for the last hour at 20 °C. Cells
were then transferred to 37 °C for various times in the continued
presence of anti-Tfn-R
gold before cooling to 4 °C.
Gold-loaded endosomes were then isolated as described above. Tfn-R and
alkaline phosphatase were immunoprecipitated from an aliquot of the
post-nuclear supernatant and from the endosome fraction. The proportion
of newly synthesized Tfn-R and alkaline phosphatase in the endosome
fraction was determined by quantitating the radioactivity in the
immunoprecipitates as described above. To determine the proportion of
total Tfn-R in the endosome fraction and hence the endosome yield, the
immunoprecipitated Tfn-R were Western blotted with H68.4, and antibody
binding was detected by chemiluminescence (ECL, Amersham, UK) and
quantitated using a PhosphorImager.
Figure 1:
Traffic of secretory horseradish
peroxidase from the TGN to the cell surface. To measure the rate at
which horseradish peroxidase is cleared from the cell HEp.2 cells
stably expressing horseradish peroxidase were incubated at 20 °C
for 4 h before transfer to 37 °C in the presence of cycloheximide.
The percent horseradish peroxidase activity cleared from the cells was
measured (squares). Results are means ± S.E. of three
observations. To determine whether horseradish peroxidase passes
through endosomes en route to the cell surface cells were incubated at
20 °C for 4 h and anti-Tfn-Rgold was added for the last hour
at 20 °C. Cells were then transferred to 37 °C in the presence
of cycloheximide and the continued presence of anti-Tfn-R
gold.
Gold-loaded endosomes were isolated and the horseradish peroxidase
activity recovered in the endosome fractions as a percent of that in
the post-nuclear supernatant was measured (circles). Results
are means ± S.E. of three
observations.
To determine whether a
significant proportion of horseradish peroxidase passes through
endosomes en route to the cell surface, cells expressing horseradish
peroxidase were incubated for 4 h at 20 °C and during the last hour
of incubation at 20 °C anti-Tfn-Rgold was added. Cells were
then transferred to 37 °C in the presence of cycloheximide and
anti-Tfn-R
gold-loaded endosomes isolated as described
previously(12) . Morphological analysis shows that the entire
Tfn recycling pathway is accessible at 20 °C, and neither the yield
nor the form of the endosomes changes significantly at this reduced
temperature. As shown in Fig. 1less than 5% of the post-nuclear
supernatant horseradish peroxidase activity was recovered in the
anti-Tfn-R
gold-isolated endosomes at any chase time studied (Fig. 1). Cells incubated as above were also examined by
electron microscopy. In conventional thin sections the distribution of
anti-Tfn-R
gold at the end of the 20 °C incubation is seen in
large (0.2-0.5 µm diameter) vacuoles and smaller (50-80
nm diameter) vesicles but is not found in the flattened cisternae of
the Golgi stack or their associated vacuoles labeled with horseradish
peroxidase (Fig. 2). Sections up to 1 µm thick were also
examined to determine whether there were continuities between
structures labeled with horseradish peroxidase and those containing
anti-Tfn-R
gold which would not be evident in thin sections. As
shown in Fig. 3, although components containing the two tracers
are closely associated double-labeled elements were not observed. We
conclude, therefore, that TGN components are unlikely to be present in
the anti-Tfn-R
gold-loaded fractions. In a previously published
study (10) , we showed that if accumulation of horseradish
peroxidase activity in Golgi elements at 20 °C is followed by
transfer to 37 °C, the horseradish peroxidase and
anti-Tfn-R
gold tracers remain distributed in separate elements.
Figure 2:
Thin section electron microscopy of the
intracellular distribution of secretory horseradish peroxidase and the
transferrin receptor. Conventional thin section (70 nm thick) of HEp.2
cells stably expressing horseradish peroxidase incubated for 3 h at 20
°C and then incubated for 1 h at 20 °C with
anti-Tfn-Rgold. Reaction product for horseradish peroxidase is
contained within Golgi cisternae and their associated vesicles. The
anti-Tfn-R
gold labels large vacuolar endosomes and small vesicles (arrowed) in the vicinity. Bar, 0.2
µm.
Figure 3: Thick section electron microscopy of the intracellular distribution of secretory horseradish peroxidase and the transferrin receptor. Thick sections (1.0 µm) of HEp.2 cells stably expressing horseradish peroxidase and incubated as in Fig. 2. Thick sections demonstrate the extensive continuity which exists between Golgi complex elements and show how closely associated they can be with endocytic elements containing gold tracer. However, even in sections which are up to 10 times thicker than the diameter of the 70-120 nm diameter vesicles and tubules the tracers are seen to be distributed in separate elements. Large arrows point to Golgi stack; arrowheads indicate gold-labeled vesicles; MVE, multivesicular endosomes; C, centriole. Bars, 0.2 µm.
Compared to the content of pre-existing Tfn-R in the endosome fraction the content of newly synthesized Tfn-R in the endosomes isolated from 20 °C incubated cells is low (Fig. 4a). This is consistent with the expectation that most of the newly synthesized Tfn-R will be retained in the Golgi at this temperature. On transfer to 37 °C, there is a rapid increase in the content of newly synthesized Tfn-R in the endosome fraction. This increase, which reaches a peak in the first 10 min is then followed by a decline over the next 10 min. The amount of newly synthesized alkaline phosphatase in endosome fractions from cells incubated at 20 °C is low and does not show any detectable increase when cells are shifted to 37 °C.
Figure 4:
Kinetics of appearance of newly
synthesized protein in endosomes and on the plasma membrane. a, to measure the rate of appearance of newly synthesized
Tfn-R in endosomes HEp.2 cells were pulse labeled with trans-S-label for 15 min and chased for 4 h at 20
°C. Anti-Tfn-R
gold was added for the last hour at 20 °C
before transfer to 37 °C in the continued presence of
anti-Tfn-R/gold. Gold-loaded endosomes were then isolated. The amount
of newly synthesized Tfn-R (squares) and alkaline phosphatase (circles) in the endosome fraction was measured by
immunoprecipitation and the total amount of Tfn-R (triangles)
in the endosome fraction was measured by Western blotting. Results are
those of a single experiment where measurement of all these parameters
was performed on the same cells. b, to measure the rate of
appearance of newly synthesized Tfn-R at the cell surface HEp.2 cells
were pulse labeled with trans-
S-label for 15 min
at 37 °C and then chased for 4 h at 20 °C before transfer to 37
°C. The amount of Tfn-R at the cell surface was determined by
measuring the proportion of newly synthesized Tfn-R accessible to
cleavage with trypsin at 4 °C (triangles). Results are
means ± S.E. of three observations. The rate of appearance of
newly synthesized Tfn-R (squares) and alkaline phosphatase (circles) in endosomes was measured as described in Fig. 4a. Results are means ± S.E. of four
observations.
To determine whether newly
synthesized Tfn-R reach the endosome before they can be detected on the
cell surface, the rate at which newly synthesized Tfn-R and alkaline
phosphatase are transported from the Golgi to the plasma membrane was
measured. Cells were pulsed for 15 min at 37 °C with trans-S-label and incubated in chase medium at 20
°C for 4 h. Cells were then transferred to 37 °C. The amount of
newly synthesized Tfn-R appearing on the cell surface was determined by
incubating the cells with trypsin at 4 °C. Under these conditions
the extracellular domain of the Tfn-R is cleaved and can be
immunoprecipitated with more than 80% efficiency(5) . The
amount of newly synthesized alkaline phosphatase appearing on the cell
surface was determined by incubating cells with
phosphatidylinositol-specific phospholipase C at 4 °C. This enzyme
cleaves the labeled alkaline phosphatase which was then
immunoprecipitated.
As shown in Fig. 4b, there is a steady increase in the amount of newly synthesized Tfn-R on the cell surface from 10 min at 37 °C when less than 5% can be detected to 30 min when, as shown by more extended incubations, a steady state of 30% is reached. Thus the rise in the amount of newly synthesized Tfn-R in endosomes at 10 min occurs before significant amounts of newly synthesized Tfn-R can be detected at the cell surface. It is noteworthy that the decline in the amount of newly synthesized Tfn-R in endosomes that occurs at 20 min is coincident with the rise in newly synthesized Tfn-R at the cell surface. Although, as shown in Fig. 4b, a small amount (6%) of newly synthesized Tfn-R is found in the endosome at the end of the 20 °C incubation, there is no indication that a similar leakage occurs to the cell surface at this temperature.
Newly synthesized alkaline phosphatase reaches the cell surface with similar kinetics to newly synthesized Tfn-R (results not shown).
A previous cell fractionation study on the hepatocyte cell
line HepG2 by Stoorvogel et al.(15) found a
significant colocalization of a secretory protein with an endocytosed
Tfn-horseradish peroxidase conjugate, and their correlative
morphological analysis suggested this was due to the presence of
endocytosed Tfn tracer in the trans-Golgi reticulum. In the
present study on HEp.2, an epithelioid cell line, we have shown that
the anti-Tfn-Rgold tracer used to load the endosomes does not
enter the TGN. This is apparent both from the cell fractionation work,
which shows that fractions containing endocytosed anti-Tfn-R
gold
contain very low levels of horseradish peroxidase after a 20 °C
block, and from the electron microscopy, which shows horseradish
peroxidase accumulates in the TGN at 20 °C but remains separate
from gold-loaded endosomes. Endosomes loaded with anti-Tfn-R
gold
contain extremely low levels of newly synthesized alkaline phosphatase
as well as horseradish peroxidase at 20 °C, and no increase is seen
on transfer to 37 °C when the newly synthesized proteins transfer
to the cell surface. We conclude, therefore, that in HEp.2 cells routes
for constitutive secretion do not pass through gold-loaded endosomes.
However, since our previous studies (12) showed by electron
microscopy that endosome fractions obtained by this gold-mediated
density shift protocol are composed primarily of the vacuolar elements
of the Tfn-R recycling pathway we cannot exclude the possibility that
these soluble phase and GPI-anchored proteins may be transferred to the
cell surface via a later step in the recycling pathway. They could for
example be transferred via the 60-nm tubules in the pericentriolar area
which form the most distal compartment in the Tfn-R recycling pathway
in these cells(16) , since these tubules are not well
represented in our density shifted fractions(12) . The
demonstration that horseradish peroxidase expressed from transfected
cDNA is efficiently cleared from the cell supports the view that this
fluid-phase tracer moves directly to the surface from the TGN because a
fluid-phase tracer entering the vacuolar elements of the endocytic
pathway would be more likely to move to the lysosome than recycle to
the cell surface(3) .
Our data demonstrate that the endosome
content of newly synthesized Tfn-R increases approximately 3-fold when
the cells are shifted from 20 to 37 °C. No increases were seen with
either horseradish peroxidase or alkaline phosphatase which provide
rigorous internal controls for the isolation procedure. The content of
newly synthesized Tfn-R within the purified endosome fraction peaks 10
min after temperature shift at 15% of the total radiolabeled Tfn-R. It
is important to emphasize that this percentage is a considerable
underestimate of the endosome content because the purification
procedure recovers only about 30% of the endosomal Tfn-R. This estimate
is based on the percentage of total Tfn-R in the endosome fraction
(approximately 20%), the earlier demonstration that the endosome
fraction provides a 150-200-fold purification containing less
than 1% plasma membrane(12) , and the percentage (70%) of total
Tfn-R that are intracellular (assayed by stripping cells incubated to
steady state with I-Tfn with low pH at 4 °C).
Correcting for the loss of endosome elements during fractionation
suggests that the endosome compartment as a whole could contain more
than three times (70/20) the amount of newly synthesized Tfn-R we
detect in purified fractions.
The efficiency with which newly synthesized Tfn-R are delivered to gold-loaded endosomes compared to fluid-phase horseradish peroxidase and GPI-linked alkaline phosphatase suggests that it is likely to be a signal dependent process. The only known trafficking signal carried by Tfn-R is the well characterized tyrosine-based internalization motif which is recognized by the selection mechanism of the clathrin-coated pits on the plasma membrane(3) . There is no evidence that this motif is recognized by the sorting mechanisms located within the clathrin lattices of the TGN, although other signals such as the dileucine signal of the CD3y subunit of the T cell antigen receptor(17) , and the signals of the invariant chain (18) which are recognized in the TGN are also known to be recognized by the clathrin lattices of the plasma membrane. It would not be very surprising therefore to find that the internalization signal carried by the Tfn-R is recognized in the TGN so that this protein like the CD3y chain and invariant chain are then sorted directly into the endosome compartment.
The suggestion that the internalization signal in the cytoplasmic domain of a predominantly cell surface protein like the Tfn-R is also recognized by clathrin lattices in the trans-Golgi has important implications for trafficking in polarized cells since it implies proteins carrying a plasma membrane internalization signal and routed through the TGN could be routed through the endosome. If this is so, then a means of ensuring that proteins trafficking through the endosome are transferred to the appropriate surface domain would also be required. In the proteins studied thus far this requirement has been satisfied. Thus in the polymeric IgA receptor which has a very efficient plasma membrane-coated pit signal there is also a signal which prevents transfer from the endosome to the apical surface(19) . Similarly in the low density lipoprotein receptor there is a proximal basolateral targetting signal which overlaps with the well characterized plasma membrane coated pit signal, as well as a distal signal, independent of the internalization signal, which prevents apical transfer(20, 21) . Interestingly, removal of the segment of the cytoplasmic domain of Tfn-R which includes the tyrosine-containing internalization signal is reported to have no influence on its delivery to the basolateral surface(22) . However, while the YTRF sequence in the Tfn-R has been shown to be all that is required for internalization in coated pits(23) , site-directed mutagenesis has shown that other regions of the cytoplasmic domain can also promote coated pit-mediated uptake at the plasma membrane(24) .
The proposal that closely related or identical signals may be recognized with varying affinity by clathrin-coated domains in both the plasma membrane and the TGN(3) , together with the knowledge that interaction with clathrin-coated domains at both sites results in delivery to the endosome, gives the endosome a central role in the selective routing of membrane proteins. It may be that the initial site of sorting on the biosynthetic pathway is the TGN and that it is the mechanisms for sorting which reside within the endosome which are primarily responsible for the delivery of proteins to the plasma membrane, the lysosome, the trans-Golgi and other intracellular destinations, such as the synaptic vesicle(25, 26) . The number and nature of the sorting mechanisms within the endosome responsible for these decisions has yet to be determined, but they probably rely upon interactions other than those which operate within clathrin-coated domains.