(Received for publication, May 11, 1995; and in revised form, September 22, 1995)
From the
A mutant influenza virus hemagglutinin, HA+8, having a
carboxyl-terminal extension of 8 amino acids that included 4 aromatic
residues, was internalized within 2 min of arriving at the cell surface
and was degraded quickly by a process that was inhibited by ammonium
chloride. Through second-site mutagenesis, the internalization sequence
of HA+8 was found to closely resemble the internalization signals
of the transferrin receptor or large mannose 6-phosphate receptor.
Comparison of the intracellular traffic of HA+8 and a series of
other HA mutants that differed in their rates of internalization
revealed a relation between the amount of the protein on the plasma
membrane at steady state and the internalization rate that would be
predicted if most of each protein recycled to the cell surface.
However, there was no simple correlation between the internalization
rate and the rate of degradation, indicating that transport to the
compartment where degradation occurred was not simply a function of the
concentration of the proteins in early endosomes. The internal
populations of both HA+8, which was degraded with a t of 1.9 h, and HA-Y543, which was degraded with
a t
of 2.9 h, were found by cell fractionation
and density-shift experiments to reside in early endosomes with little
accumulation in lysosomes. A fluid-phase marker reached lysosomes
3-4-fold faster than these proteins were degraded. Degradation of
these mutant HAs involved a rate-determining step in early endosomes
that was sensitive to some feature of the protein that depended upon
sequence differences in the cytoplasmic domain unrelated to the
internalization signal.
Cells receive nutrients, respond to the extracellular environment, and continually remodel their plasma membranes through the process of endocytosis. The different functions of endocytosis require that internalized proteins and lipids be sorted and delivered to different intracellular destinations. However, with the exception of the initial recognition of some proteins by components of clathrin-coated pits(1) , little is currently known about the mechanism by which various membrane proteins are recognized and sorted in the endocytic pathway. Following internalization, released ligands and fluid rapidly separate from the bulk of endocytosed membrane, most of which returns rapidly to the plasma membrane from which it came(2, 3, 4) . At least initially, this recycling membrane appears in the form of tubules with limited internal volume(5, 6, 7, 8) . Sorting of proteins in the fluid phase from membrane bound receptors can be explained by an iterative process of separating recycling membranes, which have a small volume and large surface area, from vesicles traveling farther along the endocytic pathway(3) . However, the sorting of membrane-bound proteins is more complex, and currently cannot be explained by a single, simple mechanism.
The majority of plasma membrane proteins recycle after internalization (9, 10) , and mutations that drastically inhibit the internalization of receptors have no effect upon the ability of the mutant proteins to recycle(11, 12) . In addition, endocytosed receptors (13, 14) and lipids return to the surface at the same rate, suggesting that recycling of membrane proteins to the cell surface is non-selective (4) . It is likely that most plasma membrane proteins are degraded in lysosomes(15) , an observation most easily explained if transport to lysosomes is also by a nonselective transport pathway. Delivery to lysosomes cannot be completely nonselective, however, since the rates of delivery to lysosomes can differ dramatically among different proteins(16) , which suggests that some proteins are specifically sorted to lysosomes. Thus, the relative contributions early in the endocytic pathway of specific recognition systems and of stochastic events that depend primarily upon protein concentration is currently uncertain, and this is particularly true for our understanding of the delivery of proteins to lysosomes.
As an
approach to understanding the features by which membrane proteins
interact with the cellular sorting machinery in the endocytic pathway,
we have used site-directed mutagenesis to construct gain-of-function
mutant proteins with altered patterns of intracellular transport. We
have introduced changes into a protein, the influenza virus
hemagglutinin (HA), ()that is highly mobile in the plasma
membrane (17) but is internalized much slower than the rate of
bulk internalization of membrane through coated pits. We have
identified a series of mutant HAs that are internalized at very
different rates. One, HA-Y543, has a single amino acid change in the
cytoplasmic domain 4 amino acids from the carboxyl terminus, a
substitution of tyrosine for cysteine at position 543(18) .
When expressed in amounts that do not saturate the internalization
capacity of CV-1 (19) or Madin-Darby canine kidney
cells(20) , this protein is internalized as rapidly as some
well characterized cell surface receptors (21, 22, 23, 24) and interacts
dynamically with coated pits(17) . We have identified several
other mutant HAs that are internalized much faster than HA-Y543. One of
these, HA+8, is internalized at 50-70%/min and binds
essentially irreversibly to coated pits(25) . HA+8 is
present only transiently in the plasma membrane and is degraded
rapidly. The amino acid sequence that contributes the internalization
signal of HA+8 is quite similar to those of the mannose
6-phosphate/insulin growth factor II (Man-6-P/IGF II) receptor and the
transferrin receptor, two proteins that are not degraded rapidly. To
investigate the possibility that HA+8 contains information
specifying rapid degradation, we compared the endocytic transport of
HA-Y543, HA+8, and second-site mutants of both proteins. Our
observations suggest that the rapid degradation of HA+8 is not
primarily determined by its rate of internalization and are consistent
with the interpretation that a rate-limiting step in degradation of
mutant HAs is a specific recognition event that controls their transfer
from early endosomes to later endocytic compartments that are probably
lysosomes. Changes in the HA cytoplasmic sequences can influence this
event independently of the internalization signal.
To truncate the sequence encoding the 8-amino acid carboxyl-terminal extension of the HA+8 mutant, the gene encoding HA+8 was subcloned into mp18 and the codon TCA encoding Ser-544 was changed to the termination codon TGA by oligonucleotide-directed mutagenesis(30) . The protein expressed from this mutant HA gene contained 4 additional amino acids at the carboxyl terminus and was named HA+4. Second site mutants of HA+8 were made by oligonucleotide directed mutagenesis on the same HA+8 template, and are named after the position and amino acid substitution made. Construction of the HA-Y543 mutant and second-site mutants of that protein has been previously reported(18, 31) . Wild type HA is referred to as HA wt. When referred to collectively, HA wt and HA mutants are called HAs.
Routine examination by immunofluorescence microscopy of CV-1 monkey fibroblasts infected for 24 h with a recombinant SV40 virus expressing HA+8 indicated that the changes in the HA+8 cytoplasmic domain had a profound effect on the steady-state distribution of the protein. Very little HA+8 was observed on the cell surface, with considerable punctate labeling of small vesicles (data not shown). After longer periods of infection when protein expression levels had risen dramatically, a significant amount of HA+8 was detected on the cell surface, an expression pattern similar to that observed for lysosomal membrane proteins expressed from recombinant vectors(36, 37, 38) . To determine if the vesicles containing HA+8 were derived from the endocytic pathway, cells expressing HA+8 or a second mutant HA that we have previously characterized, HA-Y543, were treated with cycloheximide for 2 or 6 h to allow the proteins to leave the exocytic pathway and then the cells were prepared for immunofluorescence (Fig. 1a). HA-Y543 has an internalization signal and undergoes internalization and recycling, with 60-80% of the protein at the cell surface and 20-40% internal at steady-state(18, 19, 31) . As expected, HA-Y543 was present both at the cell surface and in intracellular vesicles distributed throughout the cytoplasm, and this pattern of location did not change during the 6-h treatment with cycloheximide, although the amount of HA-Y543 detected was observed to decrease (Fig. 1a, panel B). In contrast, HA+8 exhibited very little surface labeling after 2 h of treatment with cycloheximide, but was present in vesicles located throughout the cytoplasm (Fig. 1a, panel C). After 6 h in cycloheximide, this pattern had changed. Significantly less HA+8 was detected and most of the protein was located in vesicles close to the nucleus (Fig. 1a, panel D). The loss of HA+8 from the cells was inhibited when 100 µM chloroquine was included in the medium during the final 3 h of the 6-h treatment with cycloheximide (data not shown). Clearly, HA+8 was either failing to reach the plasma membrane or was being efficiently removed from the plasma membrane by endocytosis, and appeared to be degraded faster than HA-Y543 by a process sensitive to chloroquine.
Figure 1: Immunofluorescent labeling of HA-Y543 and HA+8. Panel a, Cells expressing HA-Y543 (panels A and B) or HA+8 (panels C and D) were treated with cycloheximide for 2 h (panels A and C) or 6 h (panels B and D). Cells were fixed, permeabilized, and labeled by indirect immunofluorescence with rabbit anti-HA antibodies. B, Tf-FITC was added to the culture medium for the last 20 min of the interval of drug treatment (which was 2 h for upper panels A, B, E, and F and 6 h for lower panels C, D, G, and H). Cells were then fixed, permeabilized, and labeled with anti-HA antibodies and a second antibody conjugated to Texas Red. Cells are shown as paired images, collected simultaneously with a dual-channel laser-scanning confocal microscope fitted with filters for FITC and Texas Red fluorescence. Panels A (TfR) and B (HA-Y543), after 2 h in cycloheximide. Panels E (TfR) and F (HA+8), after 2 h in cycloheximide. Panels C (TfR) and D (HA-Y543), after 6 h in cycoloheximide. Panels G (TfR) and H (HA+8), after 6 h in cycloheximide.
To determine if the punctate staining of cells expressing HA+8 indicated that the protein mainly localized to endocytic vesicles, cells expressing either HA+8 or HA-Y543 were treated with cycloheximide for 2 or 6 h, and for the final 20 min of treatment with the drug, Tf-FITC was included in the medium to label cellular compartments containing the TfR (Fig. 1b, panels A, C, E, and G). The cells were then fixed, and the HA proteins were localized with anti-HA antibody and a second antibody conjugated to Texas Red (panels B, D, F, and H). In Fig. 1b, paired images collected with FITC fluorescence to locate transferrin bound to its receptor (panels A, C, E, and G) and Texas Red fluorescence to locate HAs in the same cell (panels B, D, F, and H) are shown. After 2 h of drug treatment, there was extensive co-localization of both HA mutant proteins and the TfR. After 6 h in cycloheximide, HA-Y543 (panel D) continued to share compartments labeled with Tf-FITC (panel C), but many cells expressing HA+8 showed essentially no co-localization, as shown in panels G (TfR) and H (HA+8). In other cells there was some co-localization (data not shown), but much less than seen after 2 h of cycloheximide treatment. A simple interpretation of these results is that in the first hours after its synthesis, HA+8 resided in early endosomes that were in contact with recycling TfR. After several hours HA+8 moved to other compartments not occupied by the TfR, and was degraded.
To determine if transport from the ER to the Golgi complex was affected by the HA+8 mutation, we measured the rate at which HA+8 and the HA wt and HA-Y543 controls acquired resistance to digestion with endo H (Table 1). Processing of the oligosaccharides of HA+8 to the complex form resistant to endo H was delayed compared to the very rapid processing of the two control proteins, but was essentially complete, as greater than 95% of the protein was processed after a 1-h chase. Thus, the mutation in HA+8 affected either the rate of its transport to the Golgi apparatus or the rate of processing of its oligosaccharides, but did not prevent the protein from leaving the ER.
To determine the rate at which HA+8 moved from the Golgi complex to the cell surface and the fate of the protein once it arrived there, we measured the rate at which pulse-labeled HA+8 could be chased to a location in which it could be cleaved into its HA1 and HA2 subunits by trypsin added to the extracellular medium(32, 41) . HA+8, HA wt, and HA-Y543 were chased at 37 °C for times between 20 and 200 min in medium containing trypsin (Fig. 2, top panels). HA+8 gained access to trypsin present continually in the chase medium at a rate of 1.3%/min, slightly slower than the rate of either of the two control proteins (Fig. 2, graphs). This observation is consistent with the interpretation that the slower acquisition of oligosaccharides resistant to endo H by HA+8 compared to the two other HAs was due to a delay in transport to the Golgi apparatus. When trypsin was present continually, over 95% of each of the three proteins was cleaved into HA1 and HA2 after a chase of 80 min. However, when trypsin was added at 4 °C only at the end of a 80-min chase, less than 10% of HA+8 was cleaved and greater than 90% had reached a compartment inaccessible to extracellular trypsin (Fig. 2, panels marked 4 °C). In contrast, >95% of HA wt remained accessible to trypsin at the cell surface and, at steady state, 70% of HA-Y543 was external and 30% internal (graphs, Fig. 2). The percent of HA+8 detected at the cell surface by this assay was entirely consistent with the immunofluorescence results, suggesting that HA+8 was only transiently at the cell surface. However, since trypsin in the medium at 37 °C was internalized and was present in the early endosome at a concentration equal to that in the extracellular medium, this assay did not distinguish between the internalization of HA+8 that had been delivered to the plasma membrane and a potential intracellular pathway transporting HA+8 to early endosomes in which trypsin was active. To distinguish between these possibilities, we employed a pulse-chase protocol to measure both the rate and the extent to which radioactive HA+8 reached the cell surface and came into contact with trypsin added to the extracellular medium at 4 °C. At this temperature, fluid phase endocytosis is blocked (42) and trypsin was not internalized.
Figure 2: Distribution of HA proteins as a function of time after synthesis. Pulse-labeled protein was chased for the times indicated with trypsin in the medium during the chase at 37 °C or trypsin added at 4 °C after the chase. HA1 and HA2 are the tryptic cleavage products of HA0. Percent accessible = (HA1 + HA2)/(HA0 + HA1 + HA2). The graphs in the lower panel present the results of densitometry of proper exposures of the autoradiograms shown in the upper panels. The percent of total protein accessible when trypsin was present at 37 °C (triangles) is a measure of the amount that left exocytic compartments during the chase. The percent accessible when trypsin was present at 4 °C (squares) is a measure of the protein present at the plasma membrane at the end of the chase period shown. The percent of each protein present in the endocytic pathway (circles) is calculated from the difference between the percent accessible when trypsin was present at 37 °C and the percent accessible when trypsin was present at 4 °C.
To ascertain that we could detect the transient presence of HA+8 at the cell surface, the majority of a cohort of pulse-labeled HA+8 was trapped in the trans Golgi network by being chased for 120 min at 18 °C(41, 43) , then released to the cell surface in a series of brief chases at 37 °C. Cells were first incubated at 37 °C for 12 min, and then treated with trypsin at 4 °C to cleave HA+8 at the plasma membrane into HA1 and HA2. The medium containing trypsin was removed, and residual trypsin was inactivated with SBTI. A series of six 5-min chases was then initiated with fresh DMEM at 37 °C to allow more HA+8 to reach the plasma membrane. After each 5-min incubation at 37 °C, the cells were returned to 4 °C and HA+8 present on the surface was cleaved by trypsin, which was then inactivated with SBTI and removed by washing in DMEM. For each chase period, samples were placed on ice for measurement of the amount of HA+8 that had become accessible to trypsin and the chase was continued with the remaining samples. Using this procedure, active trypsin never entered the cells, eliminating the possibility that internalized trypsin gained access to, and cleaved, internal HA+8. This was verified by control samples (see ``Experimental Procedures''). After the final cycle, the proteins were immunoprecipitated and the proportion of pulse-labeled proteins that had been cleaved into HA1 and HA2 subunits was determined by PAGE and quantitative autoradiography (Fig. 3). Greater than 65% of HA+8 reached the cell surface from the trans Golgi after a chase of 42 min. Longer chases were not possible due to the condition of the cells. Parallel samples incubated at 37 °C with trypsin present continually in the medium showed an identical rate of arrival at the cell surface after release of the 18 °C block. The rate taken from the slope of the curves in Fig. 3, 1.3%/min, is the same regardless of whether the trypsin was added repeatedly at 4 °C or was present continuously at 37 °C.
Figure 3: Arrival of HA+8 at the cell surface. Cohorts of pulse-labeled HA+8 that had been trapped in the trans Golgi network by low temperature were released at 37 °C (0 min of chase) and allowed to proceed to the plasma membrane under two protocols. Cells were warmed to 37 °C for a series of as many as seven brief chases at 37 °C (closed squares). Between each chase the cells were treated with trypsin at 4 °C, so that no trypsin gained access to HA+8 inside the cell. Alternatively, trypsin was present continually in the chase medium at 37 °C to have access to all HA+8 that exited the exocytic pathway (open triangles). The amount of HA+8 that was accessible to trypsin was determined as described under ``Experimental Procedures.''
Figure 4: Endocytosis of HA+8 is extremely rapid. The percent of HA wt, HA-Y543, and HA+8 that was internalized was measured as described under ``Experimental Procedures'' and is plotted as a function of time after the shift from 4 °C to 37 °C. Squares, HA wt; triangles, HA-Y543; circles, HA+8.
Figure 5: Endocytosis of second-site mutants of HA+8. Internalization assays were performed, and data plotted, as described for figure 4.
Figure 6: HA mutants differ in their rates of degradation. Cells expressing HA proteins were pulse-labeled, and the HA proteins remaining after various intervals of chase was recovered by immunoprecipitation and quantified by phosphorimaging. Representative experiments are shown. Data points are averages of duplicate samples that differed by less than 10%.
Figure 7:
HA+8 resides in early endosomes after
internalization. Panel a, separation of endosomes from
lysosomes. Cells were allowed to internalize HRP for 5 min to label
early endosomes. Membrane fractions from these cells were separated on
Percoll gradients and collected in 20 fractions. The activities of HRP
and the lysosomal enzyme -galactosidase in each fraction were
measured. Panel b, distribution of HA+8 and HA-Y543 in
endosomal membranes. Endocytic membranes from cells expressing each
protein were separated by velocity centrifugation on Percoll gradients
and collected in 20 fractions. Each fraction was divided, and
-galactosidase activity and the amount of mutant HA recovered
were determined. Data points are the average of two experiments, and
individual values differed from the mean by less than 15%. Panel
c, density shift of light membranes containing HA+8
demonstrates that the majority of HA+8 resides in early endosomes.
Cells expressing HA+8 were allowed to internalize HRP for 5 min,
and then homogenized and reacted with diaminobenzidine and
H
O
to shift the migration of vesicles
containing HRP. Membranes were centrifuged on Percoll gradients, and
HA+8 was collected from gradient fractions by
immunoprecipitation.
Early endosomes include recycling membranes carrying proteins back to the plasma membrane and vesicles, sometimes called sorting endosomes (4) , that will deliver material to late endosomes. At least some of the HA+8 was able to recycle to the cell surface from early endosomes, although we could not measure the rate at which this occurred. To demonstrate recycling directly, we treated cells expressing HA+8 and uninfected cells with cycloheximide and chased long enough for all protein to leave the exocytic pathway, then at hourly intervals added anti-HA antibody to the culture medium for 30 min at 37 °C. The cells were then fixed, and the antibody was located by indirect immunofluorescence. Only cells expressing HA+8 took up the anti-HA antibody, and they did so even after the cells had been in cycloheximide for 4 h, a period much longer than would be required to sequester the whole population of HA+8, if the protein had not been able to return to the cell surface (data not shown). Therefore, a portion of the HA+8 present in early endosomes must have been in contact with recycling membranes.
Although we did not see an accumulation of HA+8 in lysosomes by cell fractionation, the immunofluorescence experiments suggested that HA+8 exited early endosomes before it was degraded. To eliminate the possibility that HA+8 was degraded outside the endocytic pathway (for instance, at the plasma membrane), pulse-chase experiments were performed on cells expressing HA+8 that were treated with ammonium chloride. Degradation of HA+8 was completely blocked under these conditions and after 2 h of treatment, the total amount of labeled HA+8 was more than twice that in untreated cells and the amount of HA+8 recovered from denser membrane fractions had increased from 12% to 19% (data not shown).
We have engineered a series of HA mutants that enter the endocytic pathway at extremely different rates. One of these mutants, HA+8, is internalized at one of the fastest rates reported. In a recent report (25) , we have shown that HA+8 and another rapidly internalized mutant, HA+8-S548,S554, exhibit stable association with coated pits over the expected 1-2-min life-time of a clathrin-coated pit in a fibroblast (53) in keeping with their fast internalization. All of the rapidly internalized proteins used in this study, including HA+8, had a steady-state distribution consistent with the simple situation where the concentration of protein at the cell surface, multiplied by the internalization rate, was equal to the concentration inside the cell, multiplied by an externalization, or recycling, rate of 0.08-0.1/min. We confirmed directly for HA+8 and HA-Y543 that the proteins extensively co-localized with the TfR. Most of the internal population of each of these two HA mutants was accessible to HRP taken up for 5 min from the cell culture fluid, consistent with a location for the proteins early in the endocytic pathway. In spite of residing for an extended period in early endosomes, all of the mutant HAs were degraded quickly relative to the rates reported for endocytic receptors or plasma membrane proteins(16) , although in all cases the degradation rates were orders of magnitude slower than the measured internalization or the calculated recycling rates. The rate of degradation of HAs, however, was not simply proportional to their internalization rates. This is demonstrated by the rather similar degradation rates of the mutants HA-Y543 and HA-Y543,F546, which differ in internalization rates by 5-10-fold ((31) , and this work), and the significantly slower degradation rate of HA-Y543,F546 as compared to those of HA+8 or HA+8-S548,S554, in spite of the fact that all three of these mutants are internalized at similar rates.
Degradation of these proteins was not due to changes in their external domains. HA+8, like all of the mutant HAs used in this study, was found to be a well folded, trimeric HA that was resistant to proteolysis except for the trypsin-sensitive site that links the HA1 and HA2 subunits. This suggests that the different sequences of the cytoplasmic domains of these proteins directly influenced their degradation rates.
In addition, we have shown the HA+8 mutant was efficiently transported to the cell surface. Although we could not directly demonstrate that all of the protein traveled directly to the cell surface, at least 65% did so, and the rate at which HA+8 reached the cell surface under conditions where extracellular trypsin was not internalized was the same as the rate at which all of the protein came into contact with extracellular trypsin at 37 °C. The simplest explanation for these data is that after biosynthesis, all of HA+8 was exported to the plasma membrane and entered the endocytic pathway by the same mechanism as did other HA mutants. Thus, differences in rates of degradation of HA mutants, for example, HA+8 and HA-Y543, were unlikely to be due to different pathways for entering endosomes.
The pattern of retention in endosomes for a
period prior to a slower delivery to lysosomes for degradation has been
reported for several hormone receptors. After binding ligand,
-adrenergic receptors shift from a predominantly surface
population to one that is internalized and recycles for over 1 h before
degradation is measurable(54) . In the absence of EGF, the EGF
receptor internalizes slowly and returns to the plasma membrane very
rapidly. In the presence of its ligand, the receptor internalizes more
rapidly, recycles 2-4-fold slower, and is sorted to lysosomes (12) by a saturable mechanism (55) requiring sequences
of the cytoplasmic domain between residues 945 and 991 that are
separate from the internalization signal(24) . EGF receptors
have been observed to collect in multivesicular bodies that are
continuous with tubules through which transferrin receptors recycle to
the plasma membrane(8) . It has been proposed that this is due
to a specific sorting event in which the cytoplasmic domain is bound by
cellular factors that prevent recycling. Our observations suggest that
HA+8 could participate in a similar trafficking pattern, but in a
constitutive manner. In this respect HA+8 resembles the
endothelial cell adhesion protein P-selectin. In transfected Chinese
hamster ovary cells, P-selectin is transported to the cell surface and
then rapidly degraded with a half-life of 2.3 h(16) . This
rapid transport to lysosomes requires sequences in the cytoplasmic
domain of the protein that are independent of the internalization
signal, just as we observe for mutant HAs.
In addition to the few examples of proteins that seem to have lysosomal targeting sequences, the other mechanism observed to specifically direct proteins to be degraded rapidly after internalization is to cross-link them with antibodies or multivalent ligands(53, 56, 57, 58) . We have observed that HA+8 can be isolated as higher order complexes from cells lysed with octylglucoside, but not with Triton X-100(25) . However, complexes of HA+8-S548,S554, which is degraded at a similar rate, are not detected under the same conditions; thus, at present we cannot relate the ability to form higher-order complexes with the ability of HA+8 to be sorted efficiently to a compartment for degradation. Nevertheless, since all of the HA proteins studied were degraded rapidly relative to the rates reported for other proteins, it is possible that the HA external domain has properties that contribute to rapid degradation, such as the potential to form weakly associated aggregates.
Like the proteins that appear to have distinct degradation sequences, cross-linked proteins are degraded with half-times that are severalfold longer than the period required to transport HRP (42, 51, 59) or viruses (44, 60) to lysosomes. The degradation rates reported for these proteins are an order of magnitude slower than their internalization rates. At least in the case of the Fc receptor, some recycling of the protein bound to polyvalent antibody has also been reported(61) . Thus, it is possible that these proteins, like HA+8, P-selectin and activated EGF receptors, spend a protracted period in early endosomes (perhaps multivesicular bodies) in contact with recycling membranes, and are more slowly sorted to lysosomes.
Sorting of proteins in early endosomes has been proposed to occur through exclusion of proteins from recycling membranes(62) . One mechanism for achieving this that is consistent with the observations of this report would be the formation of complexes through cross-linking the cytoplasmic domains of proteins by a cytosolic endosomal ``coat'' protein (63) that recognizes a specific sorting sequence, and/or by a cytosolic chaperone-like protein that recognizes ``denatured'' cytosolic sequences. In each case, binding to the coat complex would probably be dynamic, much like the binding of HA mutants to coated pits(17, 25) . Under such conditions, proteins dissociating from the endosomal coat would likely re-enter recycling membranes. The extent to which a protein having a cytosolic recognition signal for degradation is limited to endosomes or recycled would therefore depend upon its affinity for elements of the endosomal coat. Given the rates that we and others have measured for internalization, recycling and delivery to lysosomes of rapidly degraded proteins, one would predict that either the affinity of the hypothetical endosomal coat protein for plasma membrane proteins is low, or the amount of an important component of this process is limiting. The possibility that a cellular factor or ``garbage collector'' forms complexes with HA+8 by binding to its cytoplasmic domain is a current focus of our interest in this glycoprotein.
This report is dedicated to David E. Zwart, who initiated this work before his death in December, 1991.