All living cells generate structurally complex and compositionally diverse spectra of glycans and glycoconjugates, critical for organismal evolution, development, functioning, defense, and survival. Glycosyltransferases (GTs) catalyze the glycosylation reaction between activated sugar and acceptor substrate to synthesize a wide variety of glycans. GTs are distributed among more than 130 gene families and are involved in metabolic processes, signal pathways, cell wall polysaccharide biosynthesis, cell development, and growth. Glycosylation mainly takes place in the endoplasmic reticulum (ER) and Golgi, where GTs and glycosidases involved in this process are distributed to different locations of these compartments and sequentially add or cleave various sugars to synthesize the final products of glycosylation. Therefore, delivery of these enzymes to the proper locations, the glycosylation sites, in the cell is essential and involves numerous secretory pathway components. This review presents the current state of knowledge about the mechanisms of protein trafficking between ER and Golgi. It describes what is known about the primary components of protein sorting machinery and trafficking, which are recognition sites on the proteins that are important for their interaction with the critical components of this machinery.
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Translational landscape and protein biogenesis demands of the early secretory pathway in Komagataella phaffii
Abstract Background Eukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traffic, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the flux through various routes into the early secretory pathway, we quantified the global and membrane-associated translatomes of Komagataella phaffii . Results By coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantified demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway. Conclusion A subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production.
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- Award ID(s):
- 1951942
- NSF-PAR ID:
- 10252511
- Date Published:
- Journal Name:
- Microbial Cell Factories
- Volume:
- 20
- Issue:
- 1
- ISSN:
- 1475-2859
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species,
Yarrowia lipolytica , has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion inY. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme.Results By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and
$$\alpha$$ Conclusions Overall, our combined strategies not only proved effective in improving the protein production in
Yarrowia lipolytica , but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast. -
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Abstract In plants,
N ‐acylethanolamines (NAEs) are most abundant in desiccated seeds and their levels decline during germination and early seedling establishment. However, endogenous NAE levels rise in seedlings when ABA or environmental stress is applied, and this results in an inhibition of further seedling development. When the most abundant, polyunsaturated NAEs of linoleic acid (18:2) and linolenic acid (18:3) were exogenously applied, seedling development was affected in an organ‐specific manner. NAE 18:2 primarily affected primary root elongation and NAE 18:3 primarily affected cotyledon greening and expansion and overall seedling growth. The molecular components and signaling mechanisms involved in this pathway are not well understood. In addition, the bifurcating nature of this pathway provides a unique system in which to study the spatial aspects and interaction of these lipid‐specific and organ‐targeted signaling pathways. Using whole transcriptome sequencing (RNA‐seq) and differential expression analysis, we identified early (1–3 hr) transcriptional changes induced by the exogenous treatment of NAE 18:2 and NAE 18:3 in cotyledons, roots, and seedlings. These two treatments led to a significant enrichment in ABA‐response and chitin‐response genes in organs where the treatments led to changes in development. InArabidopsis seedlings, NAE 18:2 treatment led to the repression of genes involved in cell wall biogenesis and organization in roots and seedlings. In addition, cotyledons, roots, and seedlings treated with NAE 18:3 also showed a decrease in transcripts that encode proteins involved in growth processes. NAE 18:3 also led to changes in the abundance of transcripts involved in the modulation of chlorophyll biosynthesis and catabolism in cotyledons. Overall, NAE 18:2 and NAE 18:3 treatment led to lipid‐type and organ‐specific gene expression changes that include overlapping and non‐overlapping gene sets. These data will provide future, rich opportunities to examine the genetic pathways involved in transducing early signals into downstream physiological changes in seedling growth. -
Mitochondrial and plastid functions depend on coordinated expression of proteins encoded by genomic compartments that have radical differences in copy number of organellar and nuclear genomes. In polyploids, doubling of the nuclear genome may add challenges to maintaining balanced expression of proteins involved in cytonuclear interactions. Here, we use ribo-depleted RNA sequencing (RNA-seq) to analyze transcript abundance for nuclear and organellar genomes in leaf tissue from four different polyploid angiosperms and their close diploid relatives. We find that even though plastid genomes contain <1% of the number of genes in the nuclear genome, they generate the majority (69.9 to 82.3%) of messenger RNA (mRNA) transcripts in the cell. Mitochondrial genes are responsible for a much smaller percentage (1.3 to 3.7%) of the leaf mRNA pool but still produce much higher transcript abundances per gene compared to nuclear genome. Nuclear genes encoding proteins that functionally interact with mitochondrial or plastid gene products exhibit mRNA expression levels that are consistently more than 10-fold lower than their organellar counterparts, indicating an extreme cytonuclear imbalance at the RNA level despite the predominance of equimolar interactions at the protein level. Nevertheless, interacting nuclear and organellar genes show strongly correlated transcript abundances across functional categories, suggesting that the observed mRNA stoichiometric imbalance does not preclude coordination of cytonuclear expression. Finally, we show that nuclear genome doubling does not alter the cytonuclear expression ratios observed in diploid relatives in consistent or systematic ways, indicating that successful polyploid plants are able to compensate for cytonuclear perturbations associated with nuclear genome doubling.more » « less
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Fidelity of protein targeting is essential for the proper biogenesis and functioning of organelles. Unlike replication, transcription and translation processes, in which multiple mechanisms to recognize and reject noncognate substrates are established in energetic and molecular detail, the mechanisms by which cells achieve a high fidelity in protein localization remain incompletely understood. Signal recognition particle (SRP), a conserved pathway to mediate the localization of membrane and secretory proteins to the appropriate cellular membrane, provides a paradigm to understand the molecular basis of protein localization in the cell. In this chapter, we review recent progress in deciphering the molecular mechanisms and substrate selection of the mammalian SRP pathway, with an emphasis on the key role of the cotranslational chaperone NAC in preventing protein mistargeting to the ER and in ensuring the organelle specificity of protein localization.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1186/s12934-020-01489-9
