Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway.

Immunoelectron microscopy of Saccharomyces cerevisiae cells embedded in Lowicryl K4M has been used to localize invertase and plasma membrane (PM) ATPase in secretory organelles. sec mutant cells incubated at 37 degrees C were prepared for electron microscopy, and thin sections were incubated with polyclonal antibodies, followed by decoration with protein A-gold. Specific labeling of invertase was seen in the lumen of the endoplasmic reticulum, Golgi apparatus, and secretory vesicles in mutant cells that exaggerate these organelles. PM ATPase accumulated within the same organelles. Double-immune labeling revealed that invertase and PM ATPase colocalized in secretory vesicles. These results strengthen the view that secretion and plasma membrane assembly are biosynthetically coupled in yeast.     

Order of events in the yeast secretory pathway

The sequence of posttranslational events in the export of yeast glycoproteins has been determined with the aid of mutants that affect the secretory apparatus. Temperature-sensitive secretory mutants (sec) of S. cerevisiae, when incubated at a nonpermissive growth temperature (37°C), accumulate intracellular precursor forms of exported glycoproteins, such as invertase, and expand or amplify one or more of three different secretory organelles. Characterization of haploid double-sec-mutant strains, with regard to the structure of the accumulated invertase and the morphology of the exaggerated organelles, allows assessment of the order in which the gene products are required, the sequence of invertase maturation steps and a pathway of secretory organelles. The transitions from one organelle to the next require energy and sec gene products. One of the mutants (sec7) accumulates a different organelle depending on the concentration of glucose in the medium. In normal growth medium (2% glucose), a thermally irreversible structure, the Berkeley body, predominates; in low glucose (0.1%), Golgi structures accumulate thermoreversibly. The results are consistent with the following model. Secretory proteins enter the ER, where the initial steps of glycosylation occur. Nine or more sec gene products and energy are required to transfer material to a Golgi-like structure, where further glycosylation occurs. Two or more functions and energy are required to package nearly fully glycosylated proteins into vesicles that are then transported into the bud, where they fuse with the plasma membrane in a process that requires at least ten additional gene products and energy.     

Zinc homeostasis in the secretory pathway in yeast

It is estimated that up to 10% of proteins in eukaryotes require zinc for their function. Although the majority of these proteins are located in the nucleus and cytosol, a small subset is secreted from cells or is located within an intracellular compartment. As many of these compartmentalized metalloproteins fold to their native state and bind their zinc cofactor inside an organelle, cells require mechanisms to maintain supply of zinc to these compartments even under conditions of zinc deficiency. At the same time, intracellular compartments can also be the site for storing zinc ions, which then can be mobilized when needed. In this review, we highlight insight that has been obtained from yeast models about how zinc homeostasis is maintained in the secretory pathway and vacuole.     

Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.     

Trapping parasite secretory proteins in baker's yeast

Because the function of signal sequences has been conserved during evolution it has been possible to develop both bioinformatics resources to identify them and techniques to clone genes that encode secretory proteins. The latter entail insertion of heterologous signals upstream of signal peptide deleted reporter genes. We discuss the advantages of using Saccharomyces cerevisiae for signal sequence trap technology. The yeast protein-translocation system appears to be less discriminating than that of higher eukaryotes - for example, a Theileria parva cysteine protease gene containing a recessed, nonclassical signal allows access to the secretory pathway--and yeast technology could have general application in studying elements of parasite protein trafficking.     

Export of proteins via a novel secretory pathway.

The intraerythrocytic location of the malaria parasite necessitates modification of the host cell. These alterations are mediated either directly or indirectly by parasite proteins exported to specific compartments within the host cell. However, little is known about how the parasite specifically targets proteins to locations beyond its plasma membrane. Mark Wiser, Norbert Lanners and Richard Bafford here propose an alternative secretory pathway for the export of parasite proteins into the host erythrocyte. The first step of this pathway is probably an endoplasmic reticulum (ER)-like organelle that is distinct from the normal ER. Possible mechanisms of protein trafficking in the infected erythrocyte are also discussed. The proposed ER-like organelle and alternative secretory pathway raise many questions about the cell biology of protein export and trafficking in Plasmodium.     

Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway

We have devised a genetic selection for mutant yeast cells that fail to properly deliver the vacuolar glycoprotein CPY to the lysosome-like vacuole. This has allowed us to identify mutations in eight VPL complementation groups that result in aberrant secretion of up to approximately 90% of the immunoreactive CPY. Other soluble vacuolar proteins are also affected by each vpl mutation, demonstrating that a sorting system for multiple vacuolar proteins exists in yeast. Mislocalized CPY apparently traverses late stages of the secretory pathway, since a vesicle-accumulating sec1 mutation prevents secretion of this protein. Despite the presence of abnormal membrane-enclosed organelles in some of the vpl mutants, maturation and secretion of invertase are not substantially perturbed. Thus vpl mutations define a new class of genes that encode products required for sorting of newly synthesized vacuolar proteins from secretory proteins during their transit through the yeast secretory pathway.     

The early secretory pathway contributes to autophagy in yeast

Autophagy is a starvation response in eukaryotes by which the cell delivers cytoplasmic components to the vacuole for degradation, and is mediated by a double membrane structure called the autophagosome. We have previously proposed that the specific combination of COPII like components, including Sec24p, is required for autophagy (Ishihara, N. et al. (2001) Mol. Biol. Cell, 12: 3690-3702). The autophagic defect in sec24 deleted mutant cells was, however, suppressed upon the recovery of its secretory flow by the overexpression of its homologue, Sfb2p. We have also reported that the autophagic defect is not observed in sec13 and sec31 mutants, a phenomenon that can be explained by the fact that starvation stress suppresses the secretory defect of these mutants. These observations indicate that the active flow in the early secretory pathway plays an important role in autophagy; that is, autophagy proceeds in the presence, but not in the absence of the early secretory flow. Both autophagy and its closely related cytoplasm to vacuole-targeting (Cvt) pathway occur through a pre-autophagosomal structure (PAS), and since the PAS and the functional Cvt pathway exist in all sec mutants, the early secretory pathway must be involved specifically in autophagy, subsequent to PAS formation.     

Sorting of soluble proteins in the secretory pathway of plants

The secretory pathway of plants is a network of organelles that communicate via vesicle transport. This process involves budding on donor membranes followed by their targeting to, recognition by and fusion with the acceptor membrane. Protein sorting through the plant secretory pathway is a process that requires the specific recognition of signals by receptor molecules. For soluble proteins, recognition takes place in the lumen of the secretory pathway. The sorting receptors must mediate signal transduction across the membrane to convey the information about the presence of cargo molecules to cytosolic factors, which regulate the formation of transport vesicles. Recently, a number of key elements in this process have been identified, providing tools to study protein sorting at the molecular level.     

Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway

Events in the synthesis and processing of prepro-alpha-factor have been assessed with the aid of mutants blocked at various stages in the yeast secretory pathway. In normal cells treated with tunicamycin, a precursor accumulates which is identical in molecular weight to the primary translation product synthesized in vitro. At the restrictive temperature in a mutant blocked early in the pathway (sec53), a molecule of similar molecular weight accumulates. In mutants affecting translocation into (sec59) and passage from (sec 18) the endoplasmic reticulum, a glycosylated form of the precursor containing three N-linked core oligosaccharides accumulates; however, it appears that the signal peptide is not removed. The glycosylated precursor first experiences proteolytic processing when accumulated in a mutant (sec7) blocked at the stage of the Golgi apparatus. Substantially greater amounts of the mature pheromone are seen in mutants that accumulate secretory vesicles (sec1, sec2, sec3, sec5).     

Non-invasive topology analysis of membrane proteins in the secretory pathway

We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for At ERD2 with six transmembrane domains.     

Molecular sorting of proteins into the cisternal secretory pathway

Cotranslational translocation of exportable proteins across the RER membrane prior to their release into the extracellular space has been essentially described by use of canine pancreatic microsomal membranes. Intracisternal segregation of nascent secretory proteins was observed to be irreversible and proteolytic removal of signal sequences resulted in conformationally mature and stable proteins. Structural studies on various translocation peptides from both eukaryotic and prokaryotic preparations showed that many of them have a comparable three-domain organization. A hydrophilic amino-terminal domain is followed by a core region of hydrophobic amino acids and by the region in which the proteolytic cleavage occurs. Membrane components involved in the translocation process namely the signal recognition particle and the SRP receptor as well as the way the vectorial transport mechanism of nascent secretory proteins occurs are also discussed.     

Beauty and the yeast: compartmental organization of the secretory pathway

Our perception of intracellular organelles and cellular architecture was initially based on striking light and electron micrographs of animal and plant cells. The high degree of compartmental organization within specalized mammalian secretory cells aided early efforts to track the movement of proteins through the organelles of the secretory pathway. In contrast, the morphological detail of the yeast Saccharomyces cerevisiae appeared superficially simple, even primitive, by comparison with the higher eukaryotic cells. However, the combination of genetic tools and the development of assays reconstituting vesicular traffic in yeast have facilitated the identification and characterization of individual proteins that function in the secretory pathway. Analogies between the function of yeast and mammalian proteins in vesicular traffic are being drawn with increasing frequency. In this review, the combination of genetic, biochemical, molecular and cell biological approaches used to study compartmental organization in the yeast secretory pathway will be discussed. The rapid progress in our understanding of yeast membrane traffic has revealed the beauty of working with this organism.     

RFP tags for labeling secretory pathway proteins

Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.     

Characterization of different forms of yeast acid trehalase in the secretory pathway

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec 18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.     

ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway

Resident proteins of the ER lumen carry a specific tetrapeptide signal (KDEL or HDEL) that prevents their secretion. We have previously described the isolation of yeast mutants that fail to retain such resident proteins within the cell. Here we describe ERD2, a gene required for retention. It encodes a 26 kd integral membrane protein whose abundance determines the efficiency and capacity of the retention system. Reduced expression of ERD2 leads to secretion of proteins bearing the HDEL signal, whereas overexpression of ERD2 improves retention both in wild-type cells and in other mutants. These results are consistent with other evidence that ERD2 encodes the HDEL receptor (see accompanying paper). The gene is also required, perhaps indirectly, for normal protein transport through the Golgi, and hence for growth. We discuss possible roles for ERD2 in the secretory pathway.     

Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei

We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature-sensitive secretory mutation, sec15-1. To characterize this gene further, we tested its ability to suppress other late-acting secretory mutations. The growth defect of yeast strains with sec1-1, sec1-11, sec3-2, sec6-4 and sec8-9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15-1 cells at +38 degrees C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3-101, sec5-24, sec8-9, sec10-2 and sec15-1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3-101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei.