How and when are substrates selected for type III secretion?
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Chaperone (clinical)
Secretory protein
Secretory protein
Signal recognition particle
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Budding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris. One roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally. With budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion.
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Abstract Soluble secretory proteins with a signal peptide reach the extracellular space through the endoplasmic reticulum-Golgi conventional pathway. During translation, the signal peptide is recognised by the secretory recognition particle and results in a co-translational translocation to the endoplasmic reticulum to continue the secretory pathway. However, soluble secretory proteins lacking a signal peptide are also abundant, and several unconventional (endoplasmic reticulum/Golgi independent) pathways have been proposed and some demonstrated. This work describes new features of the secretion signal called Nβ, originally identified in NaTrxh, a plant extracellular thioredoxin, that does not possess an orthodox signal peptide. We provide evidence that other proteins, including thioredoxins type h , with similar sequences are also signal peptide-lacking secretory proteins. To be a secretion signal, positions 5, 8 and 9 must contain neutral residues –a negative residue in position 9 in animal proteins– to maintain the Nβ motif negatively charged and a hydrophilic profile. Moreover, our results suggest that the NaTrxh translocation to the endoplasmic reticulum occurs as a post-translational event. Finally, the Nβ motif sequence at the N- or C-terminus could be a feature that may help to predict protein localisation, mainly in plant and animal proteins.
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Signal recognition particle
Target peptide
Protein Sorting Signals
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Budding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris. One roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally. With budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion.
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Summary In our previous study, we serendipitously discovered that protein secretion in the methylotrophic yeast Pichia pastoris is enhanced by a mutation (V50A) in the mating factor alpha (MFα) prepro‐leader signal derived from Saccharomyces cerevisiae . In the present study, we investigated 20 single‐amino‐acid substitutions, including V50A, located within the MFα signal peptide, indicating that V50A and several single mutations alone provided significant increase in production of the secreted proteins. In addition to hydrophobicity index analysis, both an unfolded protein response (UPR) biosensor analysis and a microscopic observation showed a clear difference on the levels of UPR induction and mis‐sorting of secretory protein into vacuoles among the wild‐type and mutated MFα signal peptides. This work demonstrates the importance of avoiding entry of secretory proteins into the intracellular protein degradation pathways, an observation that is expected to contribute to the engineering of strains with increased production of recombinant secreted proteins.
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Pichia
Protein Degradation
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Signal peptidase
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Secretory protein
Signal recognition particle
Signal peptidase
Transport protein
Polysome
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Abstract Much progress has been made in recent years regarding the mechanisms of targeting of secretory proteins to, and across, the endoplasmic reticulum (ER) membrane. Many of the cellular components involved in mediating translocation across this bilayer have been identified and characterized. Polypeptide domains of secretory proteins, termed signal peptides, have been shown to be necessary, and in most cases sufficient, for entry of preproteins into the lumen of the ER. These NH 2 ‐ terminal segments appear to serve multiple roles in targeting and translocation. The structural features which mediate their multiple functions are currently the subject of intense study.
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Sec61
Protein Sorting Signals
Transport protein
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Secretory protein
Sequence (biology)
Signal peptidase
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Summary The major phosphate‐repressible acid phosphatase (APase) of Saccharomyces cerevisiae , a cell wall glycoprotein, has been extensively used as a reporter protein to analyse successive steps in the yeast secretory pathway. In contrast to other yeast secretory proteins, APase can still be translocated into the endoplasmic reticulum (ER) even when it is made without its signal peptide. This property illustrates the permissiveness of targeting to the ER in yeast. Studies on APase‐containing hybrid proteins have provided some of the evidence that specific soluble factors must interact with secretory proteins prior to their translocation across the ER membrane. A systematic analysis of mutations affecting the sequence of the APase signal peptide cleavage site demonstrated that cleavage occurs only when the last amino acid of the signal sequence is small and neutral. This was one of the first studies to verify the requirements for signal peptidase cleavage that had previously only been predicted from statistical analysis. Studies performed either with inhibitors of glycosylation or with mutant APases demonstrated the critical role of core glycosylation for APase folding, which is essential for efficient transport beyond the ER. Following the fate of particular modified APases along the secretory pathway provided insights into some general properties of the secretory apparatus and illustrated the specific requirements for a given protein during its intracellular traffic.
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Signal peptidase
Transport protein
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