Surface coats and secretory trafficking in African trypanosomes

Recent advances in transfection technology have been exploited to address fundamental questions relating to secretory trafficking in African trypanosomes. Targeted gene disruptions and ectopic expression of the major stage-specific surface proteins have provided unexpected insights into both the function and assembly of the essential parasite surface coats. A growing list of novel secretory cargo molecules, as well as advances in the characterization of trypanosomal secretory machinery, provide a unique model system for the study of eukaryotic secretory processes.     

Assembling the components of the quorum sensing pathway in African trypanosomes

African trypanosomes, parasites that cause human sleeping sickness, undergo a density‐dependent differentiation in the bloodstream of their mammalian hosts. This process is driven by a released parasite‐derived factor that causes parasites to accumulate in G1 and become quiescent. This is accompanied by morphological transformation to ‘stumpy’ forms that are adapted to survival and further development when taken up in the blood meal of tsetse flies, the vector for trypanosomiasis. Although the soluble signal driving differentiation to stumpy forms is unidentified, a recent genome‐wide RNAi screen identified many of the intracellular signalling and effector molecules required for the response to this signal. These resemble components of nutritional starvation and quiescence pathways in other eukaryotes, suggesting that parasite development shares similarities with the adaptive quiescence of organisms such as yeasts and Dictyostelium in response to nutritional starvation and stress. Here, the trypanosome signalling pathway is discussed in the context of these conserved pathways and the possible contributions of opposing ‘slender retainer’ and ‘stumpy inducer’ arms described. As evolutionarily highly divergent eukaryotes, the organisation and conservation of this developmental pathway can provide insight into the developmental cycle of other protozoan parasites, as well as the adaptive and programmed developmental responses of all eukaryotic cells.     

Biogenesis of gamma-secretase early in the secretory pathway

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ₄₂, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.     

Traffic COPs of the early secretory pathway

Intracellular transport between the endoplasmic reticulum and Golgi compartments is mediated by coat protein complexes (COPI and COPII) that form transport vesicles and collect the desired set of cargo. Although the COPI and COPII coats are molecularly distinct, a number of mechanistic parallels appear to be emerging, most notably a general role for small guanine triphosphatases in co-ordinating coat assembly with cargo selection. A combination of morphological, biochemical, and genetic methods is revealing a very dynamic relationship between these compartments, and highlights a central role for COPs in directing traffic through the early secretory pathway. This review focuses on recent advances in molecular mechanisms underlying coated-vesicle assembly and connections with cellular structures.     

Protein quality control in the early secretory pathway

Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)-Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. 'Public' and 'private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion.     

Receptor-mediated protein transport in the early secretory pathway

Many secretory proteins are thought to rely upon transmembrane cargo receptors for efficient endoplasmic reticulum (ER)-to-Golgi transport. These receptors recognize specific cargo-encoded sorting signals. Only a few such cargo receptors have been characterized in detail, most of them in yeast. The only well-defined cargo receptor from mammalian cells, the LMAN1-MCFD2 complex, is required for the efficient secretion of coagulation factors V and VIII. Studies of this complex, coupled with recent advances in elucidating the basic machinery that mediates ER-to-Golgi transport, have provided a more-detailed picture of the mechanisms underlying receptor-mediated transport in the early secretory pathway. In addition to yeast studies, insights have also come from investigations into several inherited disorders that have recently been attributed to defects in the secretory pathway.     

Protein energetics in maturation of the early secretory pathway

The early secretory pathway (ESP) consisting of the endoplasmic reticulum (ER), pre-Golgi intermediates and the Golgi stack links protein synthesis to folding and vesicle trafficking to generate the membrane architecture of the eukaryotic cell. The fundamental principles that contribute to organization of the ESP remain largely unknown. We raise the possibility that assembly of the ESP is largely built on a foundation that is influenced by the kinetic and thermodynamic properties of the protein fold. Folding energetics may provide an adjustable platform for adaptor-dependent interactions with the transport machinery, suggesting the possibility that protein cargo energetics plays a central role in directing both trafficking patterns and global compartmental organization of the ESP. In this view, cargo energetics likely coordinates the composition and maturation of ER and Golgi compartments with the physiological state of the cell in different tissue and environmental settings.     

Combinatorial selection of high affinity RNA ligands to live African trypanosomes

African trypanosomiasis is a parasitic disease caused by a specific class of protozoan organisms. The best-studied representative of that group is Trypanosoma brucei which is transmitted by tsetse flies and multiplies in the blood of many mammals. Trypanosomes evade the immune system by altering their surface structure which is dominated by a layer of a variant surface glycoprotein (VSG). Although invariant surface proteins exist, they are inaccessible to the humoral immune response. Using a combinatorial selection method in conjunction with live trypanosomes as the binding target, we show that short RNA ligands (aptamers) for constant surface components can be isolated. We describe the selection of three classes of RNA aptamers that crosslink to a single 42 kDa protein located within the flagellar pocket of the parasite. The RNAs associate rapidly and with high affinity. They do not discriminate between two different trypanosome VSG variant strains and, furthermore, are able to bind to other trypanosome strains not used in the selection protocol. Thus, the aptamers have the potential to function as markers on the surface of the extracellular parasite and as such they might be modified to function as novel drugs against African trypanosomiasis.     

Lectins and protein traffic early in the secretory pathway

Lectins of the early secretory pathway are involved in selective transport of newly synthesized glycoproteins from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). The most prominent cycling lectin is the mannose-binding type I membrane protein ERGIC-53 (ERGIC protein of 53 kDa), a marker for the ERGIC, which functions as a cargo receptor to facilitate export of an increasing number of glycoproteins with different characteristics from the ER. Two ERGIC-53-related proteins, VIP36 (vesicular integral membrane protein 36) and a novel ERGIC-53-like protein, ERGL, are also found in the early secretory pathway. ERGL may act as a regulator of ERGIC-53. Studies of ERGIC-53 continue to provide new insights into the organization and dynamics of the early secretory pathway. Analysis of the cycling of ERGIC-53 uncovered a complex interplay of trafficking signals and revealed novel cytoplasmic ER-export motifs that interact with COP-II coat proteins. These motifs are common to type I and polytopic membrane proteins including presenilin 1 and presenilin 2. The results support the notion that protein export from the ER is selective.     

Auxilin facilitates membrane traffic in the early secretory pathway

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER–Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.     

Gamma-secretase complex assembly within the early secretory pathway

gamma-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the beta-secretase-processed beta-amyloid precursor protein to liberate the neurotoxic amyloid beta-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the gamma-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that gamma-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state gamma-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete gamma-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid beta-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of beta-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of gamma-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the gamma-secretase complex within the ER.     

Biosynthetic protein transport through the early secretory pathway

 Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface, cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles: COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles: (1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through the Golgi stack: vesicular transport versus cisternal maturation. Accepted: 24 October 1997     

Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome

The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human alpha1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded alpha1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded alpha1-antitrypsin for degradation by the proteasome.     

Genetic exchange in African trypanosomes

African trypanosomes are important pathogens of humans and domestic animals, but little was known, until recently, of the genetic system of these parasites. Recent results demonstrate the existence of nonobligatory genetic exchange between different stocks of T. brucei. A number of models have been put forward for the mechanism of genetic exchange, including a fusion model with subsequent random loss of chromosomes and a more conventional mendelian system.     

Metabolic compartmentation in African trypanosomes

Differences between host and parasite energy metabolism are eagerly sought after as potential targets for antiparasite chemotherapy. In Kinetoplastia, the first seven steps of glycolysis are compartmented inside glycosomes, organelles that are related to the peroxisomes of higher eukaryotes. This arrangement is unique in the living world. In this review, Christine Clayton and Paul Michels discuss the implications of this unusual metabolic compartmentation for the regulation of trypanosome energy metabolism, and describe how an adequate supply of energy is maintained in different species and life cycle stages.     

The surface of the African trypanosomes

The African trypanosomes bear on the outside of their cell membrane a single 10-15 nm thick coat of a glycoprotein. This glycoprotein may differ in structure in the predominant populations of parasitemic waves found in relapsing infections. Variant Specific Glycoprotein (VSG) range in MW between 53,000-63,000 d and may have variable amounts of carbohydrate attached at one, two, or several loci. Such differences in carbohydrate content may account in part for their range in molecular size. Approximately 30 C-terminal residues demonstrate isotypy ; i.e. these regions fall into classes having similar amino acid sequence. Modest homology has been demonstrated in two VSGs of T. congolense arising in relapsing infections although comparison of many VSG show little or no obvious homology. More recently, lipid-associated forms of VSG have been described and it is believed that these forms may be transmembrane proteins. Different VSGs appear to have different amounts of the primary sequence which have alpha-helix-forming potential. In some VSG, in excess of 80% of the structure is helical as judged by both Chou-Fasman calculations and by circular dichroism. This raises the possibility that different VSG may have different folding patterns. The arrangement of VSG on the trypanosome surface probably places the basic amino acid-rich carbohydrate-bearing C-terminus of the polypeptide chain close to the membrane. There is some protein-protein association between VSGs for which (in T. evansi) the C-terminal tail is not required. The importance of VSG structure lies not only in the fact that the molecule mediates the phenomenon of antigenic variation but also in the recent observation that VSG may act on the cellular immune system to suppress the humoral immune responses of the host.     

Interorganelle communication and membrane shaping in the early secretory pathway

Biomolecules in the secretory pathway use membrane trafficking for reaching their final intracellular destination or for secretion outside the cell. This highly dynamic and multipartite process involves different organelles that communicate to one another while maintaining their identity, shape, and function. Recent studies unraveled new mechanisms of interorganelle communication that help organize the early secretory pathway. We highlight how the spatial proximity between endoplasmic reticulum (ER) exit sites and early Golgi elements provides novel means of ER–Golgi communication for ER export. We also review recent findings on how membrane contact sites between the ER and the trans-Golgi membranes can sustain anterograde traffic out of the Golgi complex.