Several enzymatic activities alter the secondary structure of the polypeptide chain during and after import. Peptidylprolyl-isomerase (PPIs/immunophilins) catalyzes the cis–trans isomerization of proline residues, and  oxidoreductases, such as protein disulfide isomerase (PDI) and Erp57, control disulfide bond formation between correct pairs of cysteine residues.

A multitude of post-translational modifications occur in the ER: N-linked glycosylation, disulfide bond formation, lipidation, hydroxylation, oligomerization, etc. N-linked glycosylation is one of the most common posttranslational protein modifications and is .initiated by transfer of a core oligosaccharide to consensus Asn-X-Ser/Thr residues in the polypeptide chain. The covalent attachment of hydrophilic oligosaccharides serves several purposes in protein folding, assembly and trafficking: first, due to the hydrophilic nature of carbohydrates, glycosylation increases the solubility of glycoproteins and defines the attachment area for the surface of the protein. Second, due to their large hydrated volume oligosaccharides shield the attachment area from surrounding proteins. Third, oligosaccharides interact with the peptide backbone and stabilize its conformation. Lastly, sequential trimming of sugar residues is monitored by a lectin machinery to report on the folding status of the protein.

The calnexin/calreticulin cycle is one arm of a complex process termed the quality-control machinery in the ER that monitors protein conformations and dictates whether a molecule is exported to the Golgi or targeted for ER-associated degradation (ERAD). Thus, proteins that have ultimately failed ER quality control are degraded to prevent their accumulation in the ER, which might either titrate out the components of the chaperone systems or form large insoluble aggregates that would be toxic to the cell. ERAD is conserved from lower eukaryotes like yeast to mammals. Both malfolded proteins and excess subunits of multimeric proteins are retrotranslocated or dislocated back into the cytosol, which appears to be similar to the translocon used to enter the ER lumen. This retro-translocation process is usually coupled with ubiquitination, which occurs at the cytosolic surface of the ER membrane. Ubiquitin (Ub) is a highly conserved small protein that is universally expressed in eukaryotic cells. Ubiquitination of substrates is a multi-step process. Thus, if improperly folded, the protein is  retrogradely translocated to the cytosol for degradation by the proteasome (see Figure 2 and also under “Protein degradation in the cell”). Puzzling is the fact that several parallel pathways appear to exist that are capable of extracting proteins from the ER and delivering them to the proteasome for destruction. Given the complexity of the system and the medical importance of this process, the detailed study of ERAD will remain an interesting topic for the next years.

Thus, in order to travel along the secretory pathway and eventually reach their appropriate cellular destinations, newly synthesized secreted and membrane-bound proteins must fold and assemble correctly. Failure to do so results in their retention in the ER and eventual degradation. The proper conformational maturation of nascent secretory pathway proteins is both aided and monitored by a number of ER chaperones and folding enzymes in the ER quality control process mentioned above. The transport of newly synthesized polypeptides that have not yet folded completely, as well as those proteins that have folded incorrectly, are retained in the ER via their interactions with the chaperones (Figure 2; see also under “Protein folding in the cell”).

Figure 2. Schematic illustration of ER-resident chaperone functions under non-stress conditions including (1) facilitating co-translational translocation; (2) helping protein folding; (3) facilitating retro-translocation and ERAD; and (4) contributing to ER luminal calcium storage.

Storage of cellular calcium in the ER    

A major function of the ER is to store calcium that is used for intracellular signaling in response to mitogenic and growth factor signal transduction pathways. Calcium is pumped into the ER via the action of ER-localized, transmembrane ATPases known as SERCA pumps in mammalian cells. In the ER, calcium is bound through both high affinity and low affinity interactions to a number of resident ER proteins including calnexin, calreticulin, GRP94, BiP, and CaBP1. Sustained ER calcium levels are essential for normal protein folding in this organelle, as drugs like thapsigargin, which interferes with the action of the ER calcium ATPase, and A23187, which depletes ER calcium, result in dramatic and rapid activation of the ER stress or unfolded protein response (UPR).  

ER stress   

In its broadest definition, stress is the response of any system to perturbations of its normal state. For a cell or organism these can be either life-enhancing changes, e.g. feeding, or life-threatening changes, e.g. starvation. To apply this definition of stress to an organelle, e.g. the ER, we have to address the following questions: what are the physiological functions of the ER and how are they perturbated? Furthermore, we have to understand how these perturbations are sensed and how signals are transduced to initiate countermeasures to restore the original state. Only parts of these questions have been answered above and will be dealt with here.

In eukaryotic cells, the ER is the first compartment in the secretory pathway. As discussed above, it is responsible for the synthesis, modification and delivery of proteins to their proper target sites within the secretory pathway and the extracellular space. All secretory proteins enter the secretory pathway through the ER. In addition, the ER is the site for the synthesis of sterols and lipids. In lower eukaryotes, a major portion of the cell wall is synthesized in the ER. Disruption of any of these processes causes ER stress. Historically, the focus is on ER stress caused by disruption of protein folding, and little is currently known about ER stress caused, for example, by aberrations in lipid metabolism, or disruption of cell wall biogenesis. Proof of principle experiments established that expression of mutant, folding-incompetent proteins causes ER stress and an ER stress response (unfolded protein response, UPR). This is the biochemical basis for many ER storage diseases, in which folding-incompetent proteins accumulate in the ER. In vivo protein folding requires a complex ER-resident protein folding machinery. Exhaustion of the capacity of this protein folding machinery by over-expression of wild-type proteins, e.g. blood coagulation factor VIII, or antithrombin III results in the accumulation of unfolded, aggregated proteins in the ER and activation of the UPR. Recently, many physiological conditions were identified in which the demand on the ER-resident protein folding machinery exceeds its capacity, e.g. differentiation of B-cells into plasma cells, a cell type highly  specialized in secretion. 

Two simple adaptive mechanisms are employed to bring the folding capacity of the ER and its unfolded protein burden into line and return the ER to its normal physiological state: (1) upregulation of the folding capacity of the ER through induction of ER-resident molecular chaperones and foldases, and an increase in the size of the ER; the transcriptional up-regulation of ER chaperones is the hallmark of the ER stress response and occurs in all eukaryotic organisms, and (2) down-regulation of the biosynthetic load of the ER through shut-off of protein synthesis on a transcriptional and translational level, and increased clearance of unfolded proteins from the ER through upregulation of ERAD.

Conformational diseases are caused by mutations altering the folding pathway or final conformation of a protein. Many conformational diseases are caused by mutations in secretory proteins and reach from metabolic diseases, e.g. diabetes, to developmental and neurological diseases, e.g. Alzheimer’s disease (see also below).  

In terms of functions of the ER chaperones during the stress response, it would appear in many cases that they do the same thing as during normal physiological conditions but perhaps more so. A major function of the ER chaperones is to promote protein folding by preventing misfolding or aggregation. During conditions of ER stress, alterations in the ER environment can profoundly affect the folding of many proteins. This includes proteins that bind to the BiP chaperone system, as well as those that bind only to the calnexin/calreticulin system. Although BiP appears to be the sole chaperone that is monitored by the cell to sense ER stress, many of the chaperones are coordinately up-regulated. Thus, the main function of the increased levels of ER chaperones is to bind to unfolded proteins, prevent them from aggregating, and to aid and monitor their refolding if normal physiological conditions are restored to the ER. If the stress is not resolved rapidly, many unfolded proteins will be targeted for ERAD as one way to decrease the load of malfolded proteins that accumulate in the ER. So conceptually, UPR and ERAD are partially overlapping means to the same end, and it makes sense for the cell to regulate ERAD during UPR. Finally, if the ER stress conditions cannot be alleviated, in order to ultimately protect the organism, presumably by eliminating unhealthy or infected cells, an apoptotic pathway is activated, which involves the ER-localized caspase-12 protein.

Thus, a vast number of studies have revealed a role for ER chaperones in contributing to or controlling all of the major functions of the ER, including translocation of nascent polypeptide chains, folding and assembly of secretory pathway proteins, monitoring the success of this operation, and identifying those proteins that fail this quality control and targeting them for degradation. Clearly these functions of ER chaperones would be even more critical during conditions of ER stress. In addition, they play a role in storing calcium in the ER, which is important in cellular signal transduction pathways, and which also may play a role in initiating apoptosis when ER stress is sustained and induces loss of calcium from the ER. Finally, the ER chaperone BiP is central to the cellular mechanisms to sense alterations in the ER. 

Genetic and biochemical dissection of the secretory pathway   

In the late 1970s the molecular mechanisms that underlie vesicular transport were elucidated by reducing vesicular transport to a set of elementary biochemical reactions: Schekman and colleagues isolated and studied temperature-sensitive "sec" mutants of the yeast Saccharomyces cerevisiae that were defective in protein secretion, while Rothman and colleagues devised an ingenious cell-free assay to measure protein transport between cisternae of the mammalian Golgi complex (in vitro reconstitution). From these studies, it was concluded that yeast and mammals share a conserved vesicular transport machinery, which can be dissected using both genetic and biochemical tools. The results have produced a detailed molecular picture of the mechanisms of trafficking in the secretory pathway and the related endocytic and vacuolar/lysosomal targeting pathways. Central to these mechanisms are the two most critical events in the lifetime of a transport vesicle, namely budding and fusion.

ER-to-Golgi transport     

Each of the transport steps connecting ER and Golgi, and the Golgi compartments is unidirectional, and energy- and GTP dependent. Small GTP-binding proteins (20-30 kDa, e.g. the rab proteins) are distributed throughout the secretory pathway with distinctive subcellular locations and regulate vesicular trafficking between the subcompartments. In addition, the traffic is regulated by integral membrane proteins (vesicular soluble NSF attachment receptors, v-SNAREs, and target t-SNAREs; NSF=N-Ethylmaleimide-sensitive factor, v=vesicular, t=target) for docking the vesicle to the membrane of the acceptor compartment and soluble proteins (NSF and SNAPs) to initiate vesicle fusion. In this vesicular model of protein transport, all anterograde (forward) and retrograde (backward) intra-Golgi steps involve transport only via vesicles. Alternatively, a nonvesicular transport mechanism may be present in which Golgi cisternae form at the cis-face of the stack, probably by VTC fusion, and then progressively mature into trans-cisternae (cisternal maturation model). In this model, cisternae (or intermittent tubular continuities) carry secretory cargo through the stack in the anterograde direction, while vesicles transport Golgi enzymes in the retrograde direction, allowing cisternal maturation to occur by progressive uptake of material from older stacks. At the TGN, the cisternae ultimately disintegrate and evolve into a collection of secretory vesicles, including immature secretory granules.  

Vesicular transport from the ER to the Golgi complex constitutes the initial step in protein secretion. So-called COPII-coated vesicles (see below) mediate the export of newly synthesized proteins from the ER, and this transport step is coupled with COPI-mediated retrograde traffic to form a transport circuit that supports the compositional asymmetry of the ER-Golgi system. Biochemical and structural studies have advanced our understanding of the mechanisms that control vesicle formation and cargo-protein capture. Recent work has highlighted the function of transitional ER regions in specifying the location of COPII budding. COPI coat components traffic primarily from the Golgi to the ER and between Golgi cisternae. COPII-coated vesicles traffic from the ER to the Golgi. COP I and COP II coat proteins thus direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells. These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles. COP II proteins are required for selective export of newly synthesized proteins from the ER. COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER. Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex. 

Selective cargo export from the ER is brought about by the budding of COPII vesicles. While the main structural components of the COPII coat have been identified and characterized, the regulatory event(s) promoting COPII vesicle biogenesis and cargo selection still remains largely unknown. COPII vesicle biogenesis from the ER is critical for the transit of all cargo molecules to the Golgi. This process depends on vesicle budding from the ER membrane through a cycle of coat polymerisation. Following scission, unknown events trigger coat disassembly and targeting of the vesicle to the Golgi.

Proteins destined for the Golgi exit the ER at specialized ER exit sites (ERES) defined by the presence of COPII coats. The close apposition of ERES to the cis-face of the Golgi stack led to the "cisternal maturation" model for Golgi biogenesis, in which new cis-cisterna form from membranes derived from the adjacent ERES. The cisterna matures as it moves in the trans-direction through the stack, by recycling components in the cis-direction. An alternative "vesicular" model proposes that each Golgi cisterna is a stable compartment, and receives cargo by vesicular traffic. The cisternal maturation model predicts that Golgi are always spatially associated with ERES. The vesicular model allows Golgi localization to be independent of ERES.

The coated vesicle budding hypothesis  

The vesicle-transport hypothesis for protein trafficking was based on early electron microscopy studies. Vesicular transport intermediates bud from a donor organelle and then fuse with an acceptor organelle. Budding intermediates were initially identified by their electron-dense ‘coats’ and were found on the plasma membrane and intracellular organelles. Three major classes of these coated vesicles have now been purified: COPI- (coatomer) and COPII-coated vesicles (where COP stands for coat protein complex) and clathrin-coated vesicles. Coat components are needed for generation of highly curved membrane areas, recruitment of cargo (and exclusion of non-cargo proteins/lipids), vesicle scission and uncoating factor recruitment.

Just as a SNARE-based fusion underpins the hypothesis of a common mechanism for all membrane fusion, so a coat-based budding hypothesis has emerged as a common theme in vesicle budding. Clathrin-coated vesicles are named after the protein that self-polymerises into a lattice around these vesicles as they bud from the plasma membrane, trans-Golgi network (TGN) and endosomes. For all clathrin-coated vesicles, clathrin is the central organiser. It concentrates cargo adaptors, leading to a diverse protein and lipid load in the forming vesicle, and its polymerisation into a curved lattice stabilises the nascent membrane bud as it forms. The main cargo adaptor proteins characterised to date are the classical adaptor protein (AP) complexes, AP1, AP2, AP3 and AP4. Most AP complexes adapt and/or link clathrin to selected membrane cargo and lipids, and they also bind accessory proteins that regulate coat assembly and disassembly.

A long-recognised commonality between COPI, COPII and many clathrin budding pathways is that small G-proteins are required for membrane recruitment. COPI components and the clathrin adaptors (AP1, AP3, AP4 and GGAs) are recruited by Arfs (ADP ribosylation factor substrates), whereas COPII depends on the Arf-related protein Sar1. COPI and COPII coats all have the equivalent of a clathrin and an adaptor subcomplex.

Vesicle budding and fusion 

Genetic and biochemical analyses of the secretory pathway have produced a detailed picture of the molecular mechanisms involved in selective cargo transport between organelles. This transport occurs by means of vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment. Vesicle budding and cargo selection are mediated by protein coats, while vesicle targeting and fusion depend on a machinery that includes the SNARE proteins. Precise regulation of these two aspects of vesicular transport ensures efficient cargo transfer while preserving organelle identity.

The vesicular transport hypothesis

The stage was set over 30 years ago by the work of George Palade and colleagues on protein secretion. This work established that newly synthesized secretory proteins pass through a series of membrane-enclosed organelles, including the ER, the Golgi complex and secretory granules, on their way to the extracellular space. Proteins destined for residence at the plasma membrane, endosomes, or lysosomes share the early stations of this pathway (i.e., the ER and the Golgi complex) with secretory proteins. Importantly, the secretory proteins are often found within small, membrane-enclosed vesicles interspersed among the major organelles of the pathway. Such observations inspired the vesicular transport hypothesis, which states that the transfer of cargo molecules between organelles of the secretory pathway is mediated by shuttling transport vesicles. According to this hypothesis, vesicles bud from a "donor" compartment ("vesicle budding") by a process that allows selective incorporation of cargo into the forming vesicles while retaining resident proteins in the donor compartment ("protein sorting"). The vesicles are subsequently targeted to a specific "acceptor" compartment ("vesicle targeting"), into which they unload their cargo upon fusion of their limiting membranes ("vesicle fusion"). An updated representation of the steps of vesicular transport is shown in Figure 3. The processes of budding and fusion are iterated at the consecutive transport steps until the cargo reaches its final destination within or outside the cell. To balance this forward movement of cargo, organelle homeostasis requires the retrieval of transport machinery components and escaped resident proteins from the acceptor compartments back to the corresponding donor compartments ("retrograde transport"), a process that is also proposed to occur by vesicular transport. All of these steps are tightly regulated and balanced so that a large amount of cargo can flow through the secretory pathway without compromising the integrity and steady-state composition of the constituent organelles.

Figure 3. Steps of vesicle budding and fusion. (1) Initiation of coat assembly. The membrane-proximal coat components (blue) are recruited to the donor compartment by binding to a membrane-associated GTPase (red) and/or to a specific phosphoinositide. Transmembrane cargo proteins and SNAREs begin to gather at the assembling coat. (2) Budding. The membrane-distal coat components (green) are added and polymerize into a mesh-like structure. Cargo becomes concentrated and membrane curvature increases. (3) Scission. The neck between the vesicle and the donor compartment is severed either by direct action of the coat or by accessory proteins. (4) Uncoating. The vesicle loses its coat due to various events including inactivation of the small GTPase, phosphoinositide hydrolysis, and the action of uncoating enzymes. Cytosolic coat proteins are then recycled for additional rounds of vesicle budding. (5) Tethering. The “naked” vesicle moves to the acceptor compartment, possibly guided by the cytoskeleton, and becomes tethered to the acceptor compartment by the combination of a GTP bound Rab and a tethering factor. (6) Docking. The v- and t-SNAREs assemble into a four-helix bundle. (7) This “trans-SNARE complex” promotes fusion of the vesicle and acceptor lipid bilayers. Cargo is transferred to the acceptor compartment, and the SNAREs are recycled.

Role of protein coats in vesicle budding and cargo selection

The budding of transport vesicles and the selective incorporation of cargo into the forming vesicles are both mediated by protein coats. These coats are supramolecular assemblies of proteins that are recruited from the cytosol to the nascent vesicles. The coats deform flat membrane patches into round buds, eventually leading to the release of coated transport vesicles. The coats also participate in cargo selection by recognizing sorting signals present in the cytosolic domains of transmembrane cargo proteins. Vesicle budding and cargo selection at different stages of the exocytic and endocytic pathways are mediated by different coats and sorting signals. The first coats to be identified and characterized contained a scaffold protein, clathrin, as their main constituent. Clathrin coats were initially assumed to participate in most, if not all, vesicular transport steps within the cell. However, later studies demonstrated that the function of these coats was restricted to post-Golgi locations including the plasma membrane, the trans-Golgi network (TGN), and endosomes. A major discovery was the existence of non-clathrin coats that mediate vesicular transport in the early secretory pathway. One of these coats, COPII, is now known to mediate export from the ER to either the ER-Golgi intermediate compartment (ERGIC) or the Golgi complex, while another coat, COPI, is involved in intra-Golgi transport and retrograde transport from the Golgi to the ER. Of the various protein coats that have been identified to date, COPII is one of the best understood. Apart from Sar1p, the subunits of the COPII coat are structurally distinct from those of the COPI and clathrin coats. The relative simplicity of COPII, as well as its unique role in ER export, have facilitated the analysis of its assembly and function.

Cargo selection

The majority of cargo proteins are actively concentrated in COPII-coated buds and vesicles prior to export from the ER. Most transmembrane cargo proteins exit the ER by binding directly to COPII, but some transmembrane and most soluble cargo proteins bind indirectly to COPII through transmembrane export receptors. Export receptors leave the ER together with their ligands, unload their cargo into the acceptor compartment, and recycle back to the ER. The sorting signals recognized by the COPII coat are found in the cytosolic domains of transmembrane cargo proteins. These signals are quite diverse. The involvement of so many different signals in the same sorting step implies the existence of either multiple binding sites on the same recognition protein or a family of recognition proteins. Both of these solutions have evolved for COPII. The diversity of signals and recognition modes explains the ability of COPII to package a wide variety of exported proteins.

Clathrin coats are considerably more complex than COPII and COPI. Clathrin and clathrin-adaptor complexes can polymerize into spherical, cage-like structures, as can COPII, indicating that these proteins have an intrinsic ability to sculpt buds and vesicles from membranes. Thus, the clathrin-adaptor complexes appear to perform the same basic functions as the COPII coats: cytosolic signal recognition and membrane deformation. However, the clathrin vesicle cycle involves additional classes of proteins that do not seem to operate during COPII vesicle formation.

Role of SNARE proteins in vesicle fusion

After a vesicle sheds its coat, it must be targeted to the appropriate acceptor compartment. The final step in a vesicle's existence is fusion with the acceptor membrane. Remarkably, the targeting and fusion reactions both rely on the same class of proteins.

The so-called SNARE hypothesis proposes that each type of transport vesicle carries a specific "v-SNARE" that binds to a cognate "t-SNARE" on the target membrane (Figure 4). This idea fits with the observations that various SNAREs localize to different intracellular compartments. SNAREs seem to perform two major functions. One function is to promote fusion itself: SNAREs form the conserved, essential core of the fusion machinery. It is likely that in all of the transport steps in the secretory and endocytic pathways, SNAREs perform the function of overcoming the energy barrier to fusion. The second major function of SNAREs is to help ensure the specificity of membrane fusion. Different v-/t-SNARE complexes form at different steps of intracellular transport. SNAREs cannot, however, be the only specificity determinants for membrane fusion because a given v-SNARE recycles and is therefore present in both anterograde and retrograde vesicles (Figure 5B). Like many biological processes, membrane fusion employs sequential, partially redundant mechanisms to achieve high fidelity.

Not surprisingly, a plethora of accessory components and regulatory reactions modulate the action of SNAREs. This modulation is important to prevent inappropriate events of SNARE complex formation. For instance, the putative Ca²⁺ sensor synaptotagmin interacts with SNAREs and promotes synaptic vesicle fusion in response to Ca²⁺ influx. Little is known about how SNAREs are targeted to specific organelles. For the few SNAREs that have been examined, targeting determinants are present in the transmembrane sequence, the cytosolic domain, or both. An important mechanism for SNARE localization is interaction with vesicle coats. For example, SNAREs involved in ER-to-Golgi transport must be packaged into COPII vesicles during ER export and then into COPI vesicles during retrieval from the Golgi.

Figure 4. Structure and function of SNAREs (A) Crystal structure of a synaptic trans- SNARE complex. (B) The SNARE cycle. A trans-SNARE complex assembles when a monomeric v-SNARE on the vesicle binds to n oligomeric t-SNARE on the target membrane, forming a stable four-helix bundle that promotes fusion. The result is a cis-SNARE complex in the fused membrane. a-SNAP binds to this complex and recruits NSF, which hydrolyzes ATP to dissociate the complex. Unpaired v-SNAREs can then be packaged again into vesicles.

The Golgi complex    

Can proteins be transported from the Golgi back to the ER?  

Golgi formation and cisternal maturation

There is much interest in understanding how the different Golgi cisternae are organized and differentiated. Two main models exist. In the “vesicular model” of protein transport (see figure above), all anterograde (forward) and retrograde (backward) intra-Golgi steps involve transport only via vesicles. Alternatively, a nonvesicular transport mechanism may be present in which Golgi cisternae form at the cis-face of the stack, probably by VTC fusion, and then progressively mature into trans-cisternae (“cisternal maturation model”; see figures on the right and below). In this model, cisternae (or intermittent tubular continuities) carry secretory cargo through the stack in the anterograde direction, while vesicles transport Golgi enzymes in the retrograde direction, allowing cisternal maturation to occur by progressive uptake of material from older stacks. At the TGN, the cisternae ultimately disintegrate and evolve into a collection of secretory vesicles, including immature secretory granules.

Golgi proteins    

Post-translational protein modifications in the Golgi complex   

Figure 5. Possible models for the spatial segregation of exocytic events in raft and nonraft domains of the plasma membrane. A) Rafts are the preferred site for all exocytic events. Fusion may occur within a raft domain as shown or alternatively, rafts may surround the fusion site. B) Exocytosis predominates in nonraft domains. C) Lipid rafts mark the site for kiss-and-run and similar transient exocytic events, whereas full fusion of the vesicle and plasma membrane occurs in nonraft domains. D) Kiss-and-run and similar types of exocytosis occur predominantly in nonraft domains, whereas full fusion of the vesicle and plasma membrane occurs in lipid rafts. E) Raft and nonraft domains may regulate the docking and fusion of specific pools of vesicles (the Figure shows one vesicle pool with blue content, the other with green content).

Endocytosis    

Cells interact with their environment at the plasma membrane and through the endocytic pathway. Clathrin-coated pits are specialized regions of the plasma membrane that function to concentrate specific integral membrane receptors. Nutrients, growth factors, viruses and toxins, and immunoglobulins are amoung the many ligands known to bind with high affinity to receptors on the cell surface. The clathrin coated-pit regions of membrane pinch off into the cell, delivering vesicles concentrated with receptors and ligands. This complex, multistep process, known as receptor-mediated endocytosis, involves numerous structural proteins and accessory factors. The figure diagrams the process of endocytic uptake of receptors into clathrin-coated vesicles at the cell surface and the subsequent recycling of involved proteins for further rounds of uptake.

Endocrine diseases linked to the secretory pathway    

A detailed understanding of the processes by which peptide hormones are produced and released, is desirable as a growing number of endocrine disorders is linked to malfunctioning transport, sorting and processing mechanisms in the secretory pathway. For instance, mutations in the thyroid prohormone thyroglobulin may lead to defective prohormone folding and assembly, reduced ER-export and an ER storage disease (hypothyroidism); see below for more examples. In patiens with familial hyperproinsulinemia, a mutation in the B-chain of proinsulin probably causes improper prohormone folding and sorting with diversion of the mutant molecule from the regulated to the constitutive pathway. Alternatively, in such patiens an altered prohormone processing site in proinsulin may occur that results in partially cleaved proinsulin intermediates. Familial neurohypophyseal diabetes insipidus may be caused by mutations in the vasopressin-neurophysin II prohormone gene leading to inefficient signal peptide cleavage, impaired ER-export or improper prohormone folding and resulting in reduced vasopressin production. Familial hypoparathyroidism is due to a mutation in the gene region encoding the signal peptide of preproparathyroid hormone. A mutated exon-intron splice junction gives rise to exon skipping during growth hormone gene expression, resulting in misfolding of the mutant growth hormone that causes Golgi fragmentation, disrupted ER-to-Golgi traffic and familial growth hormone deficiency. Another growth disorder (Laron-type dwarfism) can be caused by a mutation in the growth hormone receptor gene, leading to defective membrane expression of the receptor. A form of diabetes mellitus appears to be due to a mutant insulin receptor leading to disrupted receptor folding and transport. Finally, PC1/PC3 inactivating gene mutations lead to multiple endocrine deficits resulting from defective prohormone processing (obesity, hyperproinsulinemia, hypogonadism).