(Received for publication, May 11, 1995; and in revised form, July 6, 1995)
From the
Measurements of the lateral mobility of native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, are capable of characterizing their interactions with coated pits in live cells (Fire, E., Zwart, D. E., Roth, M. G., and Henis, Y. I.(1991) J. Cell Biol. 115, 1585-1594). To explore the dependence of these interactions on the internalization signal and the aggregation state of the protein, we have extended this approach to investigate the interactions between coated pits and several influenza hemagglutinin (HA) mutants, which differ in the internalization signals in their short cytoplasmic tails. The lack of internalization signals in the trimeric wild-type HA enables a direct comparison between specific internalization signals introduced singly in each mutant. We have selected for these studies HA mutants that showed different internalization rates and varied in their tendency to aggregate into complexes larger than trimers. Our results indicate that the mode of interaction with coated pits (transient association-dissociation versus stable entrapment) depends on the internalization signal and affects the internalization efficiency. Mutants that contain a strong internalization signal and undergo fast endocytosis were entrapped in coated pits for the entire duration of the lateral mobility measurement, suggesting stable association with (slow dissociation from) coated pits. A mutant with a suboptimal internalization signal, which was internalized 10-fold slower, exhibited transient interactions with coated pits. Both types of interactions disappeared or were significantly reduced upon disruption of the clathrin lattices under hypertonic conditions, and were modulated following the ``freezing'' of coated pits by cytosol acidification. Unlike the dependence on the cytoplasmic internalization signal, the interactions with coated pits did not depend on the aggregation state (measured by sucrose gradient centrifugation after solubilization in n-octylglucoside) of the mutants.
Receptor-mediated endocytosis plays important roles in the
recycling of membrane proteins, receptor down-regulation, and signal
termination(1, 2, 3, 4) . In many
cell types the initial step in entering the endocytic pathway is the
clustering of specific membrane proteins in plasma membrane
clathrin-coated pits(1, 5, 6, 7) . A
large body of evidence suggests that short cytoplasmic motives on
membrane proteins destined for endocytosis are recognized by
clathrin-associated proteins (by binding to AP-2, the adaptor protein
complex associated with plasma membrane coated pits), and serve as
internalization
signals(8, 9, 10, 11, 12, 13) (reviewed in Refs. 3, 4, and 14). Two distinct classes of
such signals were
identified(3, 4, 14, 15, 16) .
The first contains at least one aromatic residue, typically tyrosine,
in a tight turn conformation (17, 18, 19, 20, 21) (reviewed
in (3) and (15) ). The second is based on leucine
(LZ, where Z = L, I, V, M, C, or A), and is
thought to be involved mainly in targeting membrane proteins from the
trans-Golgi network to endosomes (22, 23) (reviewed in (16) ). There are also indications that aggregation of certain
receptors may induce their endocytosis, as suggested by the need for
dimerization to trigger the internalization of epidermal growth factor
(EGF), or Neu receptors(2) , and by the
accumulation of aggregated macrophage Fc receptors in
lysosomes(24) .
In order to characterize the interactions of membrane proteins with coated pits in intact cells, we have recently developed a method based on comparative studies of the lateral mobilities of native and specifically mutated membrane proteins and demonstrated that it can measure their interactions with coated pits at the surface of living cells(25) . In studies conducted on several cell types and several different membrane proteins, we have demonstrated that two modes of interactions with coated pits can be encountered, dynamic interactions (several association/dissociation cycles during the measurement) (25, 26) or stable association with coated pits(27) .
In view of the large variety of internalization signals and the possible contribution of additional parameters, such as higher complex formation (aggregation) to the interactions with coated pits, it was desired to explore the relationships between the mode of these interactions (transient association/dissociation versus stable association) and internalization efficiency. To investigate this issue, we applied the lateral mobility-based approach to a series of influenza hemagglutinin (HA) mutants whose cytoplasmic domain contains different internalization signals. The absence of an internalization signal in wild-type HA (HA wt), which is excluded from coated pits(9, 25) , enables a direct comparison between specific internalization signals introduced one at a time in each mutant. We have focused on HA mutants that were internalized at different rates (28) and varied in their tendency to form higher complexes (larger than the trimeric HA wt). Our results indicate that, depending on the specific internalization signal, the HA mutants can shift all the way from no interactions to stable entrapment in coated pits, and that a shift from transient to stable entrapment correlates with significantly higher internalization rates. On the other hand, microaggregation (higher complex formation) per se does not have a major effect on the interactions with coated pits.
Interactions of membrane proteins with immobile structures are capable of immobilizing them or of retarding their lateral motion(44, 45) . The plasma membrane-coated pits are essentially immobile on the FPR experimental time scale (46) and can therefore retard the lateral mobility of membrane proteins destined for endocytosis. This phenomenon can be employed to characterize the interactions between these proteins and coated pits in live cells. Using HA wt (which is excluded from coated pits) as a control, we have used this approach to demonstrate that the internalization-competent HA-Y543 point mutant exhibits dynamic interactions with coated pits(25) . The HA system is ideal for the exploration of the dependence of the mode of interactions with coated pits and the internalization rate on the internalization signal of the membrane protein, since various internalization signals can be introduced at the HA cytoplasmic tail, keeping the rest of the protein (which is devoid of any other internalization signals) unaltered. We have therefore performed lateral mobility studies on cells expressing HA mutants that differ in their cytoplasmic internalization signal and are internalized at highly different rates (28) (see Table 1). To further substantiate the involvement of coated pits in the effects measured, the experimental design also involved treatment of cells to disperse or alter the coated pit structure.
Figure 1:
Lateral diffusion of HA+8,
HA+8-S548,S554, and HA+4 relative to HA wt. CV-1 cells were
infected with the recombinant SV40 virions and grown on glass
coverslips for 36-38 h (HA wt and HA+4) or 44-46 h
(HA+8 and HA+8-S548,S554). After labeling in the cold with
anti-HA TMR-Fab` (see ``Experimental Procedures''), they were
taken for the FPR measurements, performed in HBSS/HEPES/BSA at the
indicated temperatures. Measurements were not done at 37 °C due to
the fast internalization of HA+8 and HA+8-S548,S554 at this
temperature. Each bar is the mean ± S.E. of 30-40
measurements. HA wt (blank bars), HA+8 (bars with
diagonal lines), HA+8-S549,S554 (crossed bars),
HA+4 (black bars); A, D values; B, Rvalues.
The effect of treatments
with hypertonic medium (containing sucrose or high NaCl) on the lateral
mobilities of the HA proteins is depicted in Fig. 2. The
hypertonic conditions induced a marked increase in the R values of HA+8 and
HA+8-S548,S554, raising them very close to the R
values observed for HA wt and
HA+4, essentially abolishing the differences between the R
values of the various HA mutants. Thus,
this treatment released most of the mutant HAs formerly entrapped in
coated pits, in accord with our former observation (25) that
the dynamic interactions of HA-Y543 with coated pits are abolished
under hypertonic conditions. Interestingly, at 37 °C (where the
interactions with coated pits are more pronounced), hypertonic medium
lowered the D values for HA+8 and HA+8-S548,S554
relative to HA wt or HA+4 (an effect that was somewhat more
pronounced with sucrose, but clearly evident also with NaCl). Since
reduced D values are characteristic of transient (dynamic)
interactions, this phenomenon could reflect weaker interactions (faster
dissociation rates) of these mutant HA proteins with adaptor protein
aggregates, which were reported to remain associated with the plasma
membrane following dispersal of the clathrin lattices(40) .
Figure 2:
Effect of treatment with hypertonic medium
on the lateral mobility of HA protein mutants. CV-1 cells expressing HA
wt (blank bars), HA+8 (bars with diagonal
lines), HA+8-S548,S554 (crossed bars) or HA+4 (black bars) were grown as in Fig. 1. They were treated
with hypertonic media (DMEM supplemented with either 0.45 M sucrose or 0.225 M NaCl) as described under
``Experimental Procedures.'' After labeling with anti-HA
TMR-Fab` in the hypertonic medium, FPR experiments were conducted at 22
or 37 °C as in Fig. 1, except that the hypertonic medium was
used throughout. Each bar is the mean ± S.E. of 33-66
measurements. A, D values; B, R values.
Fig. 3depicts the effects of acidification of the cytosol by
prepulsing with NHCl on the lateral mobility of the HA
mutants. Since this treatment does not disperse the coated pits but
``freezes'' them at the plasma
membrane(40, 41, 42) , it is not expected to
disrupt the interactions with coated pits, but rather to alter them. In
the case of HA-Y543, which binds transiently to coated pits in
untreated cells, this treatment resulted in stable entrapment of
HA-Y543 in the ``frozen'' coated pits, suggesting tighter
interactions (slower dissociation from the coated pit
structures)(25) . In contrast, acidification of the cytosol of
cells expressing HA+8 or HA+8-S548,S554 had little effect on
either their R
or D values at 22
°C (compare Fig. 1with Fig. 3), with the R
values remaining below those of HA wt
or HA+4. This is the result expected if HA+8 and
HA+8-S548,S554 were already stably entrapped in coated pits prior
to cytosol acidification, since if the dissociation rate is slow enough
such that no exchange occurs on the experimental time scale, a still
slower dissociation rate will not alter the observed mode of
interaction. Interestingly, the reduction in R
of these two mutants in cytosol-acidified cells was much
more pronounced at 37 °C (Fig. 3), suggesting stronger
interactions with coated pits at this elevated temperature.
Figure 3:
Effect of cytosol acidification on the
lateral mobilities of mutant HA proteins. CV-1 cells expressing HA wt (blank bars), HA+8 (bars with diagonal lines),
HA+8-S548,S554 (crossed bars) or HA+4 (black
bars) were grown as in Fig. 1and subjected to cytosol
acidification prior to labeling with anti-HA TMR-Fab`. FPR experiments
were conducted in KA/BSA buffer (see ``Experimental
Procedures'') at 22 or 37 °C. Each bar is the mean ±
S.E. of 25-40 measurements. A, D values; B, Rvalues.
Thirty-two hours
postinfection, cells expressing one of the HA proteins (HA wt, HA-Y543,
HA+8, HA+8-S548,S554, or HA+4) were subjected to a
pulse-chase protocol with TranS-label (see
``Experimental Procedures''). The cells were lysed in a
buffer containing 1% n-octylglucoside and analyzed by velocity
sedimentation on sucrose gradients made in 1% of the same detergent,
chosen since it was shown to maintain protein-protein interactions in
many cases. This choice is justified by the failure to the detect
higher complex formation (described below) when n-octylglucoside was replaced by the more disruptive
detergents Triton X-100 or Nonidet P-40 at the lysis step (followed by
analysis on sucrose gradients containing Triton X-100). A typical
experiment comparing HA+8 and HA-Y543 is shown in Fig. 4.
While HA-Y543 was detected almost entirely in fractions 10-11,
the position normal for HA trimers under the conditions employed, most
of the HA+8 migrated lower in the gradient, corresponding to the
position of larger, faster sedimenting species. The long chase time in
these experiments (120 min) causes both HA-Y543 and HA+8 (and all
the other HA mutants employed) to exit the exocytic pathway and become
distributed both at the cell surface and in internal endocytic
organelles(28) . To determine whether the aggregation occurred
to different levels at the cell surface and after internalization,
trypsin was added to the extracellular medium at 4 °C at the end of
the chase, to distinguish between HA molecules present at the cell
surface (which are cleaved by trypsin to disulfide-bonded HA1 and HA2)
and those localized intracellularly (which are inaccessible to trypsin
and remain in the HA0 form). Fig. 4demonstrates that the ratio
of (HA1 + HA2)/HA0 (cleaved/uncleaved) was the same in gradient
fractions containing larger complexes and in those containing trimeric
HA+8. This indicates that the higher HA+8 complexes contained
both proteins that were present at the cell surface at the end of the
chase, and proteins that had entered internal compartments. In
addition, there was no loss of HA+8 after trypsin treatment,
indicating that the HA+8 in the aggregated form contained well
folded HA+8 trimers.
Figure 4:
HA-Y543 and
HA+8 differ in sedimentation profiles on sucrose gradients. CV-1
cells expressing HA-Y543 or HA+8 were pulsed with
TranS-label for 15 min, chased for 120 min, and treated
with trypsin (100 µg/ml, 45 min, 4 °C). After removal of the
trypsin, the cells were incubated with DMEM containing 100 µg/ml
STI. They were then lysed in n-octylglucoside lysis buffer.
The post-nuclear supernatants from the lysed cells were centrifuged at
22 °C on 5-30% sucrose gradients containing 1% n-octylglucoside, and 20 fractions were collected. HA-Y543 and
HA+8 polypeptides immunoprecipitated from each fraction and
analyzed by SDS-PAGE and autoradiography are shown.
The average results of several such experiments (with five samples for each HA protein), performed in two separate sets, are depicted in Fig. 5and Table 2. Each set (A and B) included HA+8 and a trimeric HA protein (HA wt or HA-Y543), to enable direct comparison and exemplify the consistency of the data. It is apparent that HA-Y543 and HA+8-S548,S554 sediment almost exclusively (85% or higher) as trimers, similar to HA wt, with very low percentages migrating below the trimer peak. In contrast, only 20 and 40% of HA+8 and HA+4, respectively, sedimented as trimers, and the two mutants exhibited a strong tendency to associate into faster sedimenting complexes. These results demonstrate that the ability of an HA mutant protein to form higher complexes does not correlate with the mode of its interactions with coated pits. Thus, HA+8 and HA+4 resemble each other in the tendency to aggregate, but only the first forms stable interactions with coated pits. Comparison between HA+8 and HA+8-S548,S554 exemplifies the other extreme, where both proteins can be stably entrapped in coated pits, but only one (HA+8) can be demonstrated to form higher oligomers in n-octylglucoside/sucrose gradients. This conclusion is substantiated by the apparent lack of correlation between higher complex formation (Fig. 5, Table 2) and the ability of the HA mutant protein to undergo internalization(28) .
Figure 5:
Quantitative comparison of the
sedimentation profiles of HA wt and the various HA mutants. Cells
expressing each of the HA protein mutants were analyzed by velocity
sedimentation, immunoprecipitation and autoradiography as in Fig. 4. For each HA protein, the results of five experiments
were scanned by densitometry and averaged. Values for individual data
points differed by less than 10% of the average. The amount of protein
recovered from each gradient fraction is plotted as a percentage of the
total protein recovered. The position of trimeric HA is indicated above
the graph. A, a set of experiments performed at 18 °C
showing the profiles of HA-Y543 (), HA+8 (
), and
HA+4 (
). B, a set of experiments (22 °C)
showing the profiles of HA wt (
), HA+8 (
), and
HA+8-S548,S554 (
).
The binding of membrane proteins to structures that are
laterally immobile affects either the rate of their lateral diffusion
(measured by D) or their mobile fraction. The nature of the
effect depends on the dissociation rate of the membrane protein from
the immobile entity: labile complexes (transient interactions) induce a
reduction in D, while stable complex formation
(``permanent entrapment'' for times long relative to the
characteristic lateral diffusion time) reduces R(25, 44) (see ``Results''). The
data on the lateral mobility of the various HA mutants in untreated
cells (Fig. 1) clearly demonstrate a reduction in R
of HA+8 and HA+8-S548,S554
relative to HA wt, with no changes in their D values. No such
effect was found for HA+4, whose lateral mobility was similar to
that of HA wt. These results are compatible with the notion that
HA+8 and HA+8-S548,S554 become stably associated with
immobile structures, most likely coated pits, for the entire duration
of the measurement. The identification of these structures as coated
pits is supported by the correlation between the reduction in R
(Fig. 1) and the internalization
rates of the HA mutants (Table 1). In addition, two independent
treatments that alter the structure of coated pits, hypertonic
treatment and cytosol acidification (discussed below), also affect the
lateral mobility of those mutants that undergo internalization ( Fig. 2and 3). The mode of the interaction with coated pits
(stable versus transient) appears to depend on the
internalization signal and to affect the internalization efficiency.
Thus, HA+8 and HA+8-S548,S554, which contain a strong
internalization motif (YKSF) closely resembling that of the transferrin
receptor (Table 1), show stable interactions with coated pits and
fast internalization. HA-Y543, which has a suboptimal internalization
signal (33) , is internalized 10-fold slower than the former
mutants (Table 1), and was shown to interact transiently (without
permanent entrapment) with coated pits(25) . Finally,
HA+4, which contains a truncated form of the HA+8
internalization signal (YK), is hardly internalized (Table 1) and
has no detectable interactions with coated pits in our experiments.
The effect of dispersing the clathrin lattices by hypertonic medium
on the lateral mobilities of the HA mutants supports the role of coated
pits in retarding the mobility of the internalization-competent
mutants. This treatment released the subpopulations of HA+8 and
HA+8-S548,S554 molecules which were immobilized in coated pits at
the surface of untreated cells, raising their mobile fractions very
near the high levels measured for non-internalized HAs (HA wt and
HA+4) (Fig. 2). While the same treatment has completely
abolished the transient interactions of another mutant, HA-Y543, with
coated pits (25) , it led to lower D values for
HA+8 and HA+8-S548,S554 relative to HA wt (or HA+4) at
37 °C, suggesting that under these conditions HA+8 and
HA+8-S548,S554 experienced transient (rather than stable)
interactions with immobile structures. Since aggregates of adaptor
proteins were reported to exist in the plasma membrane even after
dispersal of the clathrin lattices by hypertonic medium(40) ,
it is possible that the stably-entrapped mutants can still interact
with them, although the interactions are modified and shift from slow
to fast dissociation (i.e. from stable to transient
interactions). Alternatively, the two mutants could still interact
stably with membrane-attached AP-2 aggregates, providing the latter are
capable of slow motion. At lower temperatures, where the interactions
with coated pits are weaker (25) ( Fig. 1and Fig. 3), the residual interactions may become too low to be
detected. The absence of a similar effect on HA-Y543, whose diffusion
is identical to that of HA wt under hypertonic conditions(25) ,
is likely due to its interactions with coated pits being transient and
weaker to begin with, becoming insignificant upon clathrin lattice
dispersal. It therefore appears that even after dispersal of the
clathrin lattices, interactions with adaptor protein aggregates may
still exist, depending on the specific internalization signal. This
notion is supported by the observation (12) that more
-adaptin was co-precipitated with EGF receptors after clathrin
lattice dispersal, and that this phenomenon was temperature-dependent,
requiring warming to 37 °C. It should be noted that limited
aggregation of HA+8 and HA+8-S548,S554 could provide an
alternative explanation to the reduced D values of these
mutants after clathrin lattice dispersal, assuming that the fraction of
aggregated proteins is preferentially localized to coated pits prior to
the disruption of the clathrin lattices. However, this possibility can
be ruled out, since of these two mutants only HA+8 forms higher
complexes (Fig. 5). Furthermore, a significant reduction in D would require formation of large aggregates, due to the very
weak dependence of the lateral diffusion of membrane proteins on
molecular size(47) . If this were the case, HA+4 (which
shows aggregation on sucrose gradients; Fig. 5) would be
expected to diffuse at a slower rate than the nonaggregating HA wt;
however, the two proteins exhibit similar D and R
values (Fig. 1).
The studies
with cytosol-acidified cells provide further support for the
involvement of coated pits in the mobility-restricting interactions and
for their dependence on temperature. Cytosol acidification alters the
structure of coated pits, ``freezing'' them at the cell
surface (40, 42) . We have formerly shown that this
causes membrane proteins that interact transiently with coated pits
(HA-Y543, and the H1 subunit of the human asialoglycoprotein receptor)
to shift from transient to stable entrapment(25, 26) .
Such a shift is not expected for HA+8 and HA+8-S548,S554,
since they are stably entrapped in coated pits even prior to the
acidification treatment. Under such conditions, one expects that the R values of these two mutants will remain
lower than those of HA wt and HA+4, and this expectation is met (Fig. 3). These experiments also provide additional evidence for
the enhancement of the interactions with coated pits at 37 °C.
While the nature of the effect mediated on mobility by interactions
with immobile structures (transient or stable) is determined by the
binding kinetics (mainly the dissociation rate relative to the lateral
diffusion rate), the extent of the effect is determined by the
thermodynamic binding parameters(25, 44) . The
reduction in R
of HA+8 and
HA+8-S548,S554 is significantly more pronounced at 37 °C (Fig. 3), indicating that their affinity to coated pits
increases at this temperature. It is also possible that the number of
binding sites for the HA mutants increases under these conditions.
Apart from the internalization signal, higher complex formation (aggregation) may also play a role in the interactions of membrane proteins with coated pits. The mutants employed in the current study enable us to explore this possibility, since the various HA mutants investigated all fold properly into protease-resistant trimers (28) and differ only in specific sequences in the cytoplasmic domain. These sequences are therefore likely to be responsible for aggregate formation in the case of HA+8 and HA+4. This aggregation is not likely to stem from instability of these mutants in detergent, because higher complexes were observed only when cells were lysed with the mild detergent n-octylglucoside but not with Triton X-100 or Nonidet P-40. Furthermore, no disulfide bonds are involved, as one might expect for aggregates formed after lysis, as is evident by the dissolution of the aggregates in Triton X-100. At this stage, we do not know whether the aggregates are comprised exclusively of HA trimers, or contain additional proteins. At any rate, our results (Fig. 5, Table 2) clearly demonstrate that aggregate formation per se is neither sufficient nor required for interactions with coated pits and the ensuing internalization. However, due to the solublization with n-octylglucoside in these studies, the possibility that some complexes (e.g. HA+8-S548,S554) are disrupted by the detergent cannot be ruled out. Finally, our results do not exclude the possibility that in some cases, such as for the EGF and Neu receptors, dimerization can be a prerequisite for endocytosis (reviewed in (2) ). However, unlike constitutively endocytosed membrane proteins, EGF or Neu receptor dimerization is also intimately involved in their activation, manifested by enhanced tyrosine kinase activity and autophosphorylation(2) ; these could well trigger their internalization, rather than the dimerization itself(27, 48) .