Biomedical subjects

P Arvan

Publications and source records attributed to P Arvan.

At least 37 records · Page 2Linked to original sources

PubMed1997

Thyroglobulin transport along the secretory pathway. Investigation of the role of molecular chaperone, GRP94, in protein export from the endoplasmic reticulum.

GRP94 serves as a molecular chaperone in the endoplasmic reticulum (ER). In normal thyrocytes, GRP94 interacts transiently with thyroglobulin (Tg), and in thyrocytes of animals suffering from congenital hypothyroid goiter with defective thyroglobulin, GRP94 and thyroglobulin associate in a protracted fashion. In order explore possible consequences of GRP94 binding, we have studied recombinant nonmutant thyroglobulin expressed in control Chinese hamster ovary (CHO) cells in comparison to that produced in CHO cells genetically manipulated for selectively increased GRP94 expression. Levels of ER chaperones other than GRP94 did not detectably differ, and thyroglobulin achieved transport competence in both kinds of CHO cells. However, increased availability of GRP94 caused the residence time of Tg in the ER to be remarkably prolonged. This was accompanied by a major increase in Tg directly associated with GRP94 and an increase in the ER pool size of Tg. Importantly, co-immunoprecipitation analysis revealed disulfide-linked Tg complexes (previously reported as an early Tg-folding intermediate) especially associated with GRP94. Indeed, non-native Tg, GRP94, and a 78-kDa protein likely to be BiP, appeared in ternary complexes. Under these conditions, GRP94 association appears directly involved in prolongation of Tg folding and export, consistent with a role in quality control in the ER.

Animals↗

PubMed1997

Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells.

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 beta-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature beta-granules, while immunolabeling for cathepsin L, but not B, persists in mature beta-granules. By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.

Animals↗

PubMed1997

Intracellular protein transport to the thyrocyte plasma membrane: potential implications for thyroid physiology.

We present a snapshot of developments in epithelial biology that may prove helpful in understanding cellular aspects of the machinery designed for the synthesis of thyroid hormones on the thyroglobulin precursor. The functional unit of the thyroid gland is the follicle, delimited by a monolayer of thyrocytes. Like the cells of most simple epithelia, thyrocytes exhibit specialization of the cell surface that confronts two different extracellular environments-apical and basolateral, which are separated by tight junctions. Specifically, the basolateral domain faces the interstitium/bloodstream, while the apical domain is in contact with the lumen that is the primary target for newly synthesized thyroglobulin secretion and also serves as a storage depot for previously secreted protein. Thyrocytes use their polarity in several important ways, such as for maintaining basolaterally located iodide uptake and T4 deiodination, as well apically located iodide efflux and iodination machinery. The mechanisms by which this organization is established, fall in large part under the more general cell biological problem of intracellular sorting and trafficking of different proteins en route to the cell surface. Nearly all exportable proteins begin their biological life after synthesis in an intracellular compartment known as the endoplasmic reticulum (ER), upon which different degrees of difficulty may be encountered during nascent polypeptide folding and initial export to the Golgi complex. In these initial stages, ER molecular chaperones can assist in monitoring protein folding and export while themselves remaining as resident proteins of the thyroid ER. After export from the ER, most subsequent sorting for protein delivery to apical or basolateral surfaces of thyrocytes occurs within another specialized intracellular compartment known as the trans-Golgi network. Targeting information encoded in secretory proteins and plasma membrane proteins can be exposed or buried at different stages along the export pathway, which is likely to account for sorting and specific delivery of different newly-synthesized proteins. Defects in either burying or exposing these structural signals, and consequent abnormalities in protein transport, may contribute to different thyroid pathologies.

Animals↗

PubMed1996

Congenital hypothyroid goiter with deficient thyroglobulin. Identification of an endoplasmic reticulum storage disease with induction of molecular chaperones.

Recent advances in understanding the molecular pathogenesis of congenital hypothyroid goiter in cog/cog mice, have raised important questions concerning the maturation of thyroglobulin (the thyroid prohormone) in certain human kindreds with congenital goiter. We have now examined affected siblings from two unrelated families that synthesize an apparently normally glycosylated, > 300 kD immunoreactive thyroglobulin, yet have a reduced quantity of intraglandular thyroglobulin and that secreted into the circulation. From thyroid tissues of the four patients, light microscopic approaches demonstrated presence of intracellular thyroglobulin despite its absence in thyroid follicle lumina, while electron microscopy indicated abnormal distention of the endoplasmic reticulum (ER). We have confirmed biochemically that most intrathyroidal thyroglobulin fails to reach the (Golgi) compartment where complex carbohydrate modification takes place. Moreover, the disease in the affected patients is associated with massive induction of specific ER molecular chaperones including the hsp90 homolog, GRP94, and the hsp70 homolog, BiP. The data suggest that these patients synthesize a mutant thyroglobulin which is defective for folding/assembly, leading to a markedly reduced ability to export the protein from the ER. Thus, these kindreds suffer from a thyroid ER storage disease, a cell biological defect phenotypically indistinguishable from that found in cog/cog mice.

Animals↗

PubMed1996

An endoplasmic reticulum storage disease causing congenital goiter with hypothyroidism.

In humans, deficient thyroglobulin (Tg, the thyroid prohormone) is an important cause of congenital hypothyroid goiter; further, homozygous mice expressing two cog/cog alleles (linked to the Tg locus) exhibit the same phenotype. Tg mutations might affect multiple different steps in thyroid hormone synthesis; however, the microscopic and biochemical phenotype tends to involve enlargement of the thyroid ER and accumulation of protein bands of M(r) < 100. To explore further the cell biology of this autosomal recessive illness, we have examined the folding and intracellular transport of newly synthesized Tg in cog/cog thyroid tissue. We find that mutant mice synthesize a full-length Tg, which appears to undergo normal N-linked glycosylation and glucose trimming. Nevertheless, in the mutant, Tg is deficient in the folding that leads to homodimerization, and there is a deficiency in the quantity of intracellular Tg transported to the distal portion of the secretory pathway. Indeed, we find that the underlying disorder in cog/cog mice is a thyroid ER storage disease, in which a temperature-sensitive Tg folding defect, in conjunction with normal ER quality control mechanisms, leads to defective Tg export. In relation to quality control, we find that the physiological response in this illness includes the specific induction of five molecular chaperones in the thyroid ER. Based on the pattern of chaperone binding, different potential roles for individual chaperones are suggested in glycoprotein folding, retention, and degradation in this ER storage disease.

Animals↗

PubMed1996

Oligomeric assembly of thrombospondin in the endoplasmic reticulum of thyroid epithelial cells.

The thyroid endoplasmic reticulum (ER) provides an environment in which conformational maturation of thyroglobulin monomers occurs with progressive dissociation from BiP (a molecular chaperone), prior to thyroglobulin dimerization. This pattern of folding is thought to represent a pathway common to many exportable polypeptides. Thyrocytes also synthesize and secrete thrombospondin, an extracellular matrix glycoprotein that forms disulfide-linked trimers. Using a monoclonal antibody recognizing the N-terminal heparin-binding domain of thrombospondin, pulse-chase/immunoprecipitation experiments indicate that this epitope forms essentially cotranslationally. Dependent upon structural information contained within the N-terminal region, thrombospondin trimers also form and are rapidly stabilized by interchain disulfide bonds in the peritranslational period. Within 30 to 60 sec, a new epitope in the mid-molecule is detected. Additional approaches (including thrombospondin dissociation from BiP-an indirect measure of conformational maturation; t1/2 approximately 20 min) independently suggest that significant folding of monomers occurs within the trimer, i.e., well after oligomerization. These later events appear rate limiting for thrombospondin export from the ER (t1/2 approximately 30 min). The results highlight plasticity in the relationship between oligomerization and specific folding events for different proteins exported from the thyroid ER.

Animals↗

PubMed1995

Intracellular transport of proinsulin in pancreatic beta-cells. Structural maturation probed by disulfide accessibility.

In pancreatic islets, formation of beta-secretory granule cores involves early proinsulin homohexamerization and subsequent insulin condensation. We examined proinsulin conformational maturation by monitoring accessibility of protein disulfide bonds. Proinsulin disulfides are intact immediately upon synthesis, but are > or = 90% sensitive to in vivo reduction with 2 mM dithiothreitol; wash out of dithiothreitol leads to reoxidation, proinsulin transport, and conversion to insulin. With t1/2 approximately 10 min, newly synthesized proinsulin becomes resistant to disulfide reduction, correlating with endoplasmic reticulum (ER) export. However, inhibition of ER export with brefeldin A blocks acquisition of resistance to reduction, and once proinsulin arrives in the Golgi, it resists reduction despite brefeldin treatment. Moreover, in vivo, resistance of proinsulin disulfides is overcome after increasing [dithiothreitol] > 10-fold, or in vitro, in islets lysed in a zinc-free, but not a zinc-containing, medium. Employing 30 mM dithiothreitol in vivo, a further decrease in disulfide accessibility is observed following proinsulin conversion to insulin. Incubation of islets with chloroquine or zinc enhances and diminishes accessibility of insulin disulfides, respectively. We hypothesize that two major conformational changes culminating in granule core formation, proinsulin hexamerization and insulin condensation, are sensitive to zinc and occur upon ER exit and arrival in immature secretory granules, respectively.

Animals↗

PubMed1995

Polarized distribution and delivery of plasma membrane proteins in thyroid follicular epithelial cells.

Thyroid follicular cells coordinate several oppositely located surface enzyme activities. Recent studies have raised questions about the basic mechanisms used to achieve thyroid surface polarity. We investigated these mechanisms in primary thyroid epithelial monolayers cultured on porous filters. In the steady state, most Na+/K(+)-ATpase and aminopeptidase N were available for surface biotinylation, and these proteins exhibited physiological distributions (basolateral and apical, respectively). Glycosylphosphatidylinositol-anchored proteins were also apically distributed. By pulse-chase, newly synthesized transmembrane proteins exhibited polarized surface delivery that was oriented similarly to that observed at steady state. Little time elapsed between acquisition of Golgi-specific processing and cell surface arrival. Interestingly, when either newly synthesized or steady state-labeled thyroid peroxidase was similarly analyzed, only approximately 30% of the enzyme was ever detected at the cell surface. Of this, the majority was localized apically. The data suggest that most thyroid peroxidase remains intracellular in these monolayers, consistent with the possibility of intracellular iodination activity in addition to apical extracellular iodination. Nevertheless, in filter-polarized thyrocytes, most newly synthesized plasma membrane proteins appear to be sorted in the Golgi complex for direct delivery to apical and basolateral domains.

Animals↗

PubMed1995

Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum.

Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or to hydrophobic surfaces of secretory proteins while they form proper disulfide bonds (Wada, I., W.-J. Ou, M.-C. Liu, and G. Scheele, J. Biol. Chem. 1994. 269:7464-7472). Because Tg has multiple disulfides, as well as glycans, we tested a brief exposure of live thyrocytes to dithiothreitol, which resulted in quantitative aggregation of nascent Tg, as analyzed by SDS-PAGE of cells lysed without further reduction. Cells lysed in the presence of dithiothreitol under non-denaturing conditions caused Tg aggregates to run as reduced monomers. For cells lysed either way, after in vivo reduction, Tg coprecipitated with calnexin. After washout of dithiothreitol, nascent Tg aggregates dissolved intracellularly and were secreted ultimately. 1 h after washout, > or = 92% of labeled Tg was found to dissociate from calnexin, while the fraction of labeled Tg bound to BiP rose from 0 to approximately 40%, demonstrating a "precursor-product" relationship. Whereas intralumenal reduction was essential for efficient Tg coprecipitation with calnexin, Tg glycosylation was not required. These data are among the first to demonstrate sequential chaperone function involved in conformational maturation of nascent secretory proteins within the ER.

Animals↗

PubMed1994

Formation of the insulin-containing secretory granule core occurs within immature beta-granules.

Contrasting with earlier immunocytochemical studies of pancreatic islet beta-cells, several recent reports describe polypeptide prohormone processing within the trans-Golgi network (TGN). Such a concern is relevant to beta-cells, wherein cleavage of the connecting polypeptide from soluble proinsulin triggers insulin condensation. Two distinct models describe how hormone condensation might enhance efficiency of storage in secretory granules: sorting for entry into granules would be favored if insulin condensed in the TGN; sorting by retention within granules would be favored if insulin condensed after proinsulin entry into immature granules (IGs). To distinguish these possibilities, we utilized Brefeldin A (BFA) to prevent protein export from the TGN. BFA did not inhibit the activity of proinsulin-processing enzymes, nor did it alter the behavior of the TGN by fractionation on Percoll density gradients. Moreover, BFA did not inhibit constitutive-like protein traffic originating from IGs. However, BFA blocked proinsulin transfer from the TGN to IGs. As long as proinsulin resided in the TGN, conversion to insulin did not occur. Washout of BFA restored granule formation and subsequent insulin production. These data provide biochemical confirmation that the generation of insulin, which is crucial for its passive condensation, occurs with IGs rather than in the TGN.

Animals↗

PubMed1994

Intracellular iodination of thyroglobulin in filter-polarized thyrocytes leads to the synthesis and basolateral secretion of thyroid hormone.

Thyroid follicles perform several functions that depend upon epithelial polarity: secretion of thyroglobulin (Tg) to the apical lumen, uptake of iodide for Tg iodination, and the manufacture of thyroid hormone for delivery to the bloodstream. In this report we examine Tg processing by thyroid epithelial monolayers cultured on porous filters. Basolateral 125I uptake resulted in thyrotropin-dependent radiolabeling of Tg in cells and apical medium. Polarized thyrocytes iodinated exogenous gamma globulins (IgG), demonstrating labeling in the apical extracellular space. Apical catalase addition inhibited the appearance of apical [125I]IgG and [125I]Tg, but had no effect on cell-associated [125I]Tg, indicating additional iodination of Tg in an intracellular compartment. A similar conclusion was drawn from radioiodination experiments at 20 degrees C. Intracellular iodination was selective for Tg forms receiving prior Golgi carbohydrate modifications. During a 2-h chase, [125I]Tg was exported from cells to apical medium, while modest amounts of thyroxine were secreted with a majority to the basolateral medium. Neither radioiodination at 20 degrees C nor apical catalase addition blocked formation or secretion of [125I]thyroxine during the chase. Thus in filter-grown thyroid epithelial cells, prior to apical extracellular iodination, intracellular iodination of Tg begins the process leading to formation of thyroxine.

Animals↗

PubMed1994

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells.

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C-peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus-dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.

Animals↗

PubMed1993

Polarized secretion of thrombospondin is opposite to thyroglobulin in thyroid epithelial cells.

In addition to thyroglobulin, primary thyrocytes secrete into the culture medium significant quantities of p500, a protein so named because of its M(r) > or = 500,000. Epithelial monolayers cultured on porous filters serve as a useful model system in which to study protein secretion. From these monolayers, thyroglobulin, the precursor in thyroid hormonogenesis, was released with apical predominance, while p500 was found mostly in the basolateral medium. Thyrocyte exposure to thyrotropin augmented selectively thyroglobulin but not p500 production. By contrast, exposure to cycloheximide actually augmented p500 production, a response observed for immediate-early proto-oncogenes. Using thyrocyte conditioned medium, the p500 protein was purified to homogeneity. Peptide sequencing of tryptic fragments of purified p500 showed identity to thrombospondin. Immunoprecipitation of thrombospondin from media bathing primary thyrocytes and the FRTL5 cell line quantitatively recovered p500, confirming its identity and indicating an epithelial origin. Gel filtration of secreted thrombospondin eluted at a high molecular weight, suggesting complexation with components of the extracellular matrix. Further, immunofluorescence showed cellular codistribution of thrombospondin and thyroglobulin, although thrombospondin exhibited predominantly an extracellular, basolateral deposition. It seems likely that thrombospondin production by thyrocytes plays a role in the growth or development of the thyroid epithelium.

Animals↗

PubMed1993

Hormonal regulation of thyroglobulin export from the endoplasmic reticulum of cultured thyrocytes.

To understand how the endoplasmic reticulum (ER) of the thyrocyte remains flexible to physiologic changes in the load of exportable proteins, we have examined hormonally-induced increments in thyroglobulin (Tg) flux and pool size in the ER, the relationship between kinetics of Tg folding and ER export, and steady-state levels of molecular chaperones. Tg production was increased > or = 5-fold by chronic exposure to thyrotropin (TSH), and > or = 25-fold by exposure to a mixture of TSH, insulin, transferrin, and hydrocortisone (4H). In TSH-grown cells Tg assembly was accelerated, specifically involving early folding intermediates that lead to a compact monomer. Accelerated dissociation of nascent Tg from the binding protein, BiP, was observed in parallel. TSH exposure was accompanied by modest increases in ER chaperones as well as accelerated Tg export from the thyrocyte ER. However, in 4H-grown thyrocytes, although there were further increases in ER chaperones, monomer maturation was slowed and the association between nascent Tg and BiP was prolonged. Nevertheless, export from the ER remained accelerated, indicating that exit from the ER must include other regulated steps that occur after the folding of exportable proteins. Thus, protein folding may not necessarily be the rate-limiting step in the export of newly synthesized proteins from the ER.

Amino Acid Sequence↗

PubMed1993

Disulfide-linked aggregation of thyroglobulin normally occurs during nascent protein folding.

In the endoplasmic reticulum (ER) of cultured porcine thyrocytes, newly synthesized thyroglobulin (Tg, the precursor in thyroid hormone synthesis) initially forms protein aggregates, which are dissolved into monomers and then assembled to dimers, before intracellular transport and secretion. However, studies suggest that in different physiological states and in different cells, folding efficiency in the ER may vary; with this in mind we have set out to further characterize the phenomenon of nascent Tg aggregation. In primary cultured thyrocytes, fresh thyroid follicular tissue (of porcine and rat origin), and the FRTL-5 cell line, nascent Tg appears transiently aggregated with mispaired, interchain disulfide linkages. Using a cell lysis procedure that maximally inhibits proteolysis as well as artifactual disulfide formation, Tg aggregates of M(r) > or = 2,000,000 can be stably isolated by gel filtration. Furthermore, stimulation with thyrotropin and other hormones that enhance Tg production may alter but does not eliminate formation of these aggregates. We conclude that transient disulfide-linked aggregation occurs normally during Tg folding in the ER of thyroid epithelial cells.

Animals↗

PubMed1992

Protein sorting and secretion granule formation in regulated secretory cells.

Formation of secretion granules in regulated secretory cells involves packaging a subject of proteins undergoing intracellular transport into specific vesicular carriers that function in stimulus-dependent exocytosis. Recent findings suggest that immature granules are a site of passive sorting, involving condensation of regulated secretory proteins. Proteins that are not condensed are stored to a lesser degree and are enriched in unstimulated, constitutive-like secretion. While these observations have helped to distinguish possible mechanisms of secretory protein sorting, there are only recent hints about the sorting processes that may be required to create the regulated secretory carrier membranes.

Journal Article↗

PubMed1992

Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment.

We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C-peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway.

Animals↗

PubMed1992

Transient aggregation of nascent thyroglobulin in the endoplasmic reticulum: relationship to the molecular chaperone, BiP.

Because of its unusual length, nascent thyroglobulin (Tg) requires a long time after translocation into the endoplasmic reticulum (ER) to assume its mature tertiary structure. Thus, Tg is an ideal molecule for the study of protein folding and export from the ER, and is an excellent potential substrate for molecular chaperones. During the first 15 min after biosynthesis, Tg is found in transient aggregates with and without interchain disulfide bonds, which precede the formation of free monomers (and ultimately dimers) within the ER. By immunoprecipitation, newly synthesized Tg was associated with the binding protein (BiP); association was maximal at the earliest chase times. Much of the Tg released from BiP by the addition of Mg-ATP was found in aggregates containing interchain disulfide bonds; other BiP-associated Tg represented non-covalent aggregates and unfolded free monomers. Importantly, the immediate precursor to Tg dimer was a compact monomer which did not associate with BiP. The average stoichiometry of BiP/Tg interaction involved nearly 10 BiP molecules per Tg molecule. Cycloheximide was used to reduced the ER concentration of Tg relative to chaperones, with subsequent removal of the drug in order to rapidly restore Tg synthesis. After this treatment, nascent Tg aggregates were no longer detectable. The data suggest a model of folding of exportable proteins in which nascent polypeptides immediately upon translocation into the ER interact with BiP. Early interaction with BiP may help in presenting nascent polypeptides to other helper molecules that catalyze folding, thereby preventing aggregation or driving aggregate dissolution in the ER.

Animals↗