Compartmentation and turnover of the low density lipoprotein receptor in skin fibroblasts.

The low density lipoprotein receptor (LDLR) was immunoprecipitated from [35S]methionine-labeled skin fibroblasts derivatized at 4 or 18 degrees C with an impermeant biotinylating reagent. Separation of derivatized and underivatized receptor from immunoprecipitates by selective binding to streptavidin-agarose allowed assessment of receptor protein cellular compartmentation and rates of intercompartmental transfer. At both 4 and 18 degrees C the amount of LDLR that is derivatized in cells labeled to near steady state saturates after 1-2 h of reaction at, respectively, 47 and 70% of total immunoprecipitable receptor protein. On the basis of temperature titration experiments, protein exposed only to the cell surface reacts at 4 degrees C; raising the temperature of biotinylation to 18 degrees C provides access to an additional pool of receptor protein. Remaining LDLR is derivatized at 37 degrees C. LDLR unreactive at 18 degrees C largely resides in membrane compartment(s) devoid of plasma membrane on the basis of its fractionation on Percoll gradients. While total cellular LDLR and 4 degrees C-derivatized LDLR labeled to steady state turn over in a first order manner (t1/2 = 12-13 h), the specific activity of pulse-labeled, 4 degrees C-accessible protein peaks after 1-2 h of chase and reaches a reduced level by 3 h of chase. These latter results show that the newly synthesized LDLR is transiently enriched at the cell surface prior to achieving equilibrium distribution between the cell surface and intracellular pools.

While total cellular LDLR and 4"Cderivatized LDLR labeled to steady state turn over in a first order manner (tIj2 = 12-13 h), the specific activity of pulse-labeled, 4'C-accessible protein peaks after l-2 h of chase and reaches a reduced level by 3 h of chase. These latter results show that the newly synthesized LDLR is transiently enriched at the cell surface prior to achieving equilibrium distribution between the cell surface and intracellular pools.
The LDLR' is a 115,000-dalton membrane glycoprotein receptor that recognizes apoproteins B and E (1). Binding of LDL apoprotein B triggers internalization of ligand and receptor and delivery of ligand to lysosomes. The receptor then recycles back to the cell surface where it can bind new ligand (2). Altered or insufficient receptor binding, synthesis, or activity results in increased serum LDL and cholesterol, an inherited disorder known as familial hypercholesterolemia (1). LDLR, like other receptors, binds ligand at the cell surface but is present both on the cell surface and other intracellular compartments. Thus, only a fraction of receptors are available to bind exogenous ligand. The localization of intracellular receptors in endosomes, lysosomes, or membrane compartments on the biosynthetic pathway has not been rigorously quantitated and may be cell-specific. Most measurements of * This work was supported by a grant from the Medical Research Foundation of Oregon. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC. Section 1734 solely to indicate this fact. receptor compartmentation distinguish only between surfaceaccessible and -inaccessible protein. These include susceptibility to extracellular proteases (3, 4), ability to bind ligand at low temperature (5, 6) or release ligand in response to acidification (7), antibody binding (8), chemical reactivity with membrane-impermeable reagents (g-11), and sensitivity to neuraminidase (12). Some of these methods can be difficult to control. Others are applicable only to receptor proteins. Moreover, most allow only steady state evaluation of membrane protein surface localization and do not permit measurement of rates of appearance and disappearance from different membrane compartments. This study reports measurements of LDLR compartmentation and intercompartmental transfer obtained by using a new type of compartmentation assay. The assay is called the biotinylation/recovery assay and combines cell surface biotinylation using a cleavable reagent with immunoprecipitation (13). In agreement with another study (9), the results identify 39-49% of the receptor that is localized to the cell surface, the rest residing within the cell. In addition, variation of the derivatization temperature distinguishes between two different intracellular compartments as well as the surface compartment. Also, newly synthesized receptor protein is found to turn over more rapidly on the cell surface than in intracellular compartments. This latter result shows that receptor transiently appears at the cell surface before it distributes to other membranes.  A, to mark cell surface proteins, cells were chemically labeled at 4°C with lz51 as described under "Materials and Methods." Cells were then broken and fractionated on 15% Percoll gradients as described under "Materials and Methods." Fractions were recovered from the bottom to the top of the tube. B, unlabeled cells were fractionated as in (A), but the lysosomal enzyme, @-glucuronidase, was assayed as described under "Materials and Methods" to identify fractions rich in this lysosomal marker. C, cells were labeled for 36 h as described under "Methods and Methods" and fractionated as in (A). The cpm precipitated by 10% trichloroacetic acid to estimate total protein and lactate dehydrogenase (LDH) to estimate cell cytosol are shown. D, cells were labeled for 36 h as described under "Materials and Methods" and fractionated as in (A). LDLR was immunoprecipitated from each fraction and then separated into streptavidin-agarose bound or unbound, resolved on SDSpolyacrylamide gels, and exposed to x-ray film. Arrows mark the position of the LDLR (Mr = 130,000) while the line marks the position of a putative degradation in product of the LDLR that apears in the unbound fraction only.

MATERIALS
2 to 50 h in the presence of excess cold methionine and LDLdepleted human serum or fetal bovine serum. In agreement with previous studies (22,23) receptor in these confluent cells was degraded at a rate that was first order and unaffected by the type of serum present in the chase medium (Fig. 1). The measured half-life of 12-13 h is identical (23) to or slightly lower (22) than that seen by others in the same cell type. Unlike the case of other membrane proteins (24), receptor degradation is not slowed by lysosomotropic amines such as methylamine (23; results not shown). Some LDLR appears to be shed into the media in a shortened form as evidenced by the appearance of an immunoprecipitated protein in the media of cells pulse-labeled and chased for 24 h but not of those chased for 1 h.* If related to LDLR, this media protein, however, accounts for only a small fraction of receptor lost between 1 and 24 h of chase. No measurable cell turnover was seen during the same time interval as detected by loss of  Table I. At temperatures >23"C, 95% or more of the receptor protein becomes streptavidin-agarose-bound. This shows that there is little receptor that cannot be derivatized, providing its access to the biotinylating reagent is not restricted by membrane fusion events. At derivatizing temperatures from 4 to 8"C, 42% or less of the immunoprecipitated LDLR is streptavidin-agarose-bound. These values in some experiments can be as low as 25%, the variability between experiments largely dependent on the method of detection. Values obtained from autoradiograms tend to be lower than those obtained by removing and counting the radioactivity directly from gels. The latter is likely to be the most accurate method of estimating differences between bound and unbound receptor pro- To distinguish biochemically between compartments accessible at 4 and 18"C, and those inaccessible at 18"C, derivatized cell homogenates were fractionated on self-forming Percoll gradients. Such gradients can separate plasma membrane from lysosomes and dense endosomes (21). I found that, without DNase treatment, fibroblast membranes tend to aggregate at the top of the gradient. DNase treatment causes mitochondria and lysosomes to shift to the bottom fractions of the gradient (19). As shown in Fig. 3, protein labeled at the cell surface with 1251 at low temperature to mark cell surface-exposed protein appeared at the top of the gradient while fractions rich in /3-glucuronidase (Fig. 3B) or N-acetylglucosaminidase (not shown) exhibited peaks both at the bottom (fraction 1) and the top (fractions 9, 10, and 11) of the gradient. This bimodal distribution of lysosome enzyme markers also seen in other studies (21,29) may be attributed to the presence of lysosomal hydrolases in light endosomes (30), two forms of lysosomes (21), damage to some lysosomes during cell homogenization, or aggregation unaffected by DNase treatment. Displacement of the peak of P-glucuronidase activity one fraction heavier than that of the plasma membrane marker peak fraction, however, suggests that those light membrane compartments enriched in this activity are denser than plasma membrane. Cells were then labeled to steady state and chased for 1 h to dilute out newly synthesized receptor that had not reached the cell surface. LDLR from cells derivatized at 4°C (not shown) or 18°C (Fig. 30) corn&rated with 1251surface-labeled protein and thus could be localized to the plasma membrane or light endosomes. LDLR underivatized at 18°C (Fig. 3D), on the other hand, was distributed throughout the gradient with peaks in fractions 1 and 10. These results show that the bulk of underivatized receptor resides in compartments other than plasma membrane. Another immunoprecipitated band slightly smaller than LDLR appears only in fraction 1. The same band can also be seen in pulsechase experiments but only after a pulse and chase of 4 h or longer (see Fig. 4). These two pieces of data argue for this band representing a proteolytically modified form of the receptor found exclusively in denser membrane compartments. Chemical characterization of the isolated polypeptides, however, would be required to substantiate this conclusion.
tein. Between derivatizing temperatures of 15 and 23"C, the percent of total receptor bound to streptavidin-agarose jumps from 49 to 95%. Between 18 and 20°C the percent of receptor derivatized is intermediate between that derivatized at lower temperatures and that derivatized at temperatures >23"C. These results taken together with previously published data suggest that cellular LDLR can be separated into three groups: that which is derivatized at 4"C, that which is not derivatized at 4°C but is at 18"C, and that which remains underivatized at 18°C. 4"Cderivatized receptor is likely to reside on the cell surface. Receptor that is derivatized at 18°C but not at 4°C resides in a compartment accessible only at 18°C. Receptor refractory to derivatization at 18°C represents receptor that is inaccessible to the derivatizing agent but still chemically reactive. In each case it is assumed that streptavidin-agarosebound receptor is derivatized while streptavidin-agarose unbound receptor is underivatized.
If temperature of derivatization serves to block access of the NHS-ss-biotin to intracellular compartments, increasing time of derivatization would be expected to saturate derivatizable receptor at any given temperature. In both the case of 4 and 18°C derivatization (Fig. 2), the amount of receptor bound to streptavidin-agarose saturates by l-2 h of reaction. As might be expected, at 4°C the amount of derivatized receptor bound to streptavidin-agarose saturates more rapidly than at 18°C; the additional time may be needed to access intracellular compartments. It is not clear in the case of 18°C derivatized receptor whether the derivatizing agent reacts with cell surface-exposed proteins which then internalize or Turnover of the LDLR in Different Membrane Compartments-If receptor in different cellular compartments can be isolated by the protocol described, the rate of appearance and stability of newly synthesized, immunoprecipitated LDLR recovered in these compartments can also be evaluated. For this purpose fibroblast cultures were pulse-labeled for 1 h with [35S]methionine and chased for different intervals before being derivatized at 4 or 18°C. Both streptavidin-agarosebound and -unbound receptor protein-specific activity was measured after each fraction was separated by SDS-polyacrylamide or agarose gel electrophoresis. When cells were derivatized at 4"C, newly synthesized LDLR appeared at highest specific activity in the bound fraction after 1 h of chase and then decreased in specific activity from 1 to 2 h, after which it remained at constant specific activity for up to 8 h (Figs. 4A and 5A). This same result was seen in four separate experiments regardless of whether pulse-labeled cells were chased in the presence of LDL-depleted human serum (Fig.  4A) or fetal bovine serum (Fig. 5A). The specific activity of unbound receptor peaked at 2 h of chase. In pulse-chase experiments a putative precursor form of the receptor is identified at slightly greater mobility (Mr = 95,000) in the unbound fraction in the absence of chase. This protein was Law Density Lipoprotein Receptor Turnover 0.0 . , . , . , . , . , .  Fig. 4. A, streptavidin-agarose-bound LDLR bands from cells pulse-labeled, chased, and then derivatized at 4°C (0-O) and from cells labeled to steady state, chased, and then derivatized at 4°C (O-0) were eluted from modified agarose gels and the radioactivity recorded as cpm. B and C, the LDLR bands from streptavidin-agarose bound (a-0) and unbound (0-O) fractions shown in autoradiograms in Fig. 3, A-C, were excised and the absorbance of eluted silver grains for each at 500 nm was recorded against a blank of the same size removed from the same lane. B, cells were pulse-labeled, chased, and treated with NHS-ss-biotin at 4°C before isolation of bound and unbound receptor. C, cells were pulse-labeled, chased, and treated with NHS-ss-biotin at 18°C before isolation of bound and unbound receptor. D, cells were labeled for 36 h before being chased and treated with NHS-ss-biotin at 4°C and isolation of bound and unbound always absent after 1 h of chase and was found to represent up to half of the unbound receptor pool in other experiments in the absence of chase. The increase in the total LDLR between 0 and 1 h of chase (Fig. 5, B and C) reflects continued conversion of precursor into mature protein, a process that takes 30 min to complete' (15).
When pulse-chased cells were derivatized at 18"C, streptavidin-agarose-bound receptor turned over more slowly than that bound after 4°C derivatization (Fig. 4B). Thus the metabolic stability of LDLR in the 4°C pool is distinctly different from that in the 18°C pool. The specific activity of receptor in the unbound fraction after 18°C derivatization peaked at 2-6 h of chase before decaying at the same rate as bound receptor. These results are expressed in a quantitative form in Fig. 5C. The putative breakdown product of the receptor, also seen on Percoll gradients, is shown in Fig. 4B, he 4, of the unbound protein.
If the transient appearance of LDLR in the 4°C pool reflects the behavior of newly synthesized protein, 4°C compartmentalized LDLR in steady state labeled cells should not exhibit the same rapid turnover as shown by pulse-labeled receptor. This is shown by the decay of radiolabeled receptor in the bound fractions from cells labeled for 36 h before a chase (Fig.  4C). Quantitative expression of these results (Fig. 5, A and D) shows that both bound and unbound receptor have about the same half-life (tl,* = 10 h). Turnover of streptavidinagarose-bound LDLR in cells derivatized at 18°C and labeled to steady state was no different from that seen in cells derivatized at 4°C (results not shown).

DISCUSSION
The biotinylation/recovery assay described herein allows the isolation of labeled protein appearing at the cell surface and measurement of its turnover. Although transiently high concentrations of newly synthesized proteins that chemically react with exogenous agents have been seen at the cell surface in other studies (10, ll), this is the first description of this cell surface behavior for a specific protein. The novel finding reported here is that a significant fraction of newly synthesized LDLR transiently localizes to the 4°C accessible compartment prior to its transfer to another compartment. Decrease in the specific activity of 4"C-accessible LDLR between 1 and 2 h of chase could be due to its release into the media (shedding) or internalization.
Although only trace amounts of shed LDLR were recovered in the media by immunoprecipitation, it cannot be excluded that shed protein loses its immunoreactivity.
On the other hand, LDLR unbound to streptavidin-agarose after derivatizing cells at 4°C briefly increased in specific activity between 1 and 2 h of chase before its subsequent decay. Receptor unbound after derivatizing cells at 18°C increased in specific activity between 2 and 6 h of chase. These results suggest movement of newly synthesized receptor from the cell surface to the 18°C accessible/ 4°C inaccessible and finally to 18°C inaccessible membrane compartments.
The distinct difference in turnover of newly synthesized, streptavidin-agarose-bound receptor between 4"C-derivatized (Fig. 5B) and 18"C-derivatized (Fig. 5C) cells and between pulse-labeled and steady state labeled 4"C-ac-cessible receptor (Fig. 50) argues for the rapid transfer of a major portion of newly synthesized receptor from the cell surface to intracellular membranes.
If the bulk of receptor that appears at the cell surface after a l-h chase is shed into the medium, l$"C-derivatized protein, which includes cellsurface protein, would also exhibit rapidly decreasing specific activity between 1 and 2 h of chase, and this is not the case. The transient appearance of newly synthesized receptors at the cell surface prior to redistributing to other compartments implies that sorting decisions to determine membrane protein compartmentation must be made after their cell surface appearance. It is likely that this redistribution of newly synthesized receptor simply reflects its constitutive recycling between the cell surface and intracellular membrane compartments (31). Whether constitutive recycling receptors are those that segregate into clathrin-coated pits when they reach the cell surface is unclear. Similar measurements on cells expressing the internalization defective LDLR mutant (32) should provide the answer to this question.
Aside from the efficacious recovery of cell-surface proteins, another feature of the biotinylation/recovery assay is that intracellular plasma membrane protein can be conveniently separated into three cellular membrane compartments defined by access of derivatizing agent at different temperatures. Although it seems most probable that proteins inaccessible at 4°C but accessible at 18°C are found in recycling endosomes, it cannot be excluded that these proteins also lie on the cell surface, but for other reasons are unreactive at the lower temperature.
While differentiation of 4-and 18°Caccessible compartments may only be possible by electron microscopy, 18%derivatized and -underivatized receptor can be biochemically distinguished on Percoll gradients. Percoll gradient separation of derivatized protein shows its co-localization with cell-surface and endosomal protein at the top of the gradient. Underivatized receptor is distributed differently on Percoll gradients than derivatized receptor. Its appearance in all gradient fractions and highest concentration in both the bottom and top fractions reflects its residence in vesicles of different density than plasma membrane. The retention of a third or less of underivatized receptor at the top of the gradient in plasma membrane-enriched fractions may have resulted from either imperfect fractionation or some plasma membrane localized receptor that remains underivatized or derivatized but not bound to the streptavidin-agarose.
Incomplete derivatization or incomplete binding of derivatized protein to streptavidin-agarose may also explain some experimental variability in compartmentation studies as well as the appearance of more streptaviclin-agarose unbound receptor in pulse-chase studies than expected (e.g. Fig. 5, B and C). For this reason it is advisable when doing compartmentation measurements to make triplicate determinations or carry out titrations as shown in Fig. 2 proteins is high as this reagent derivatizes a large number of plasma membrane proteins (11).