Internalization of Heterologous Sugar Transporters by Endogenous α-Arrestins in the Yeast Saccharomyces cerevisiae

Sen, Aditi; Acosta-Sampson, Ligia; Alvaro, Christopher G.; Ahn, Jonathan S.; Cate, J.H.D.; Thorner, Jeremy

doi:10.1128/aem.02148-16

Cited by 15 publications

(20 citation statements)

References 54 publications

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“…In fact, the yeast maltose transporter Mal21, which is resistant to glucose-induced endocytosis, showed higher maltose uptake activity than that of Mal61 (31). In addition, deletion of four arrestin-like proteins, including Rod1, increased the cellobiose fermentation of yeast expressing cellobiose transporters from the filamentous fungus Neurospora crassa (32). However, creD disruption had a negative effect on ␣-amylase production, whereas MalP was still retained at the plasma membrane in the presence of glucose (Fig.…”

Section: Discussionmentioning

confidence: 97%

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillusoryzae

Tanaka

Hiramoto

Tada

et al. 2017

Appl Environ Microbiol

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Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucoseinduced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although ␣-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on ␣-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB. The ␣-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the ␣-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae. IMPORTANCEIn eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucoseinduced endocytosis of transporters is mediated by their ubiquitination, and arrestinlike proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillus oryzae. Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB, which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved ␣-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae.

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Section: Discussionmentioning

confidence: 97%

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillusoryzae

Tanaka

Hiramoto

Tada

et al. 2017

Appl Environ Microbiol

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“…The authors also discovered that the truncation of the C-terminal tail of STP1 by 16 amino acids improved growth significantly on glucose, while no growth was seen on glucose if the truncation was extended to 32 amino acids. The intracellular parts have also been shown to be involved in substrate-induced ubiquitination and subsequent degradation of N. crassa cellodextrin transporters when heterologously expressed in yeast [ 30 ]. Analysis of the CRT1 amino acid sequence by UbPred ubiquitination site prediction server [ 31 ] gave two possible ubiquitination sites, both located in the N-terminal intracellular part of the protein.…”

Section: Discussionmentioning

confidence: 99%

Studies on sugar transporter CRT1 reveal new characteristics that are critical for cellulase induction in Trichoderma reesei

Havukainen

Valkonen

Koivuranta

et al. 2020

Biotechnol Biofuels

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Background Trichoderma reesei is an ascomycete fungus that has a tremendous capability of secreting extracellular proteins, mostly lignocellulose-degrading enzymes. Although many aspects of the biology of this organism have been unfolded, the roles of the many sugar transporters coded in its genome are still a mystery with a few exceptions. One of the most interesting sugar transporters that has thus far been discovered is the cellulose response transporter 1 (CRT1), which has been suggested to be either a sugar transporter or a sensor due to its seemingly important role in cellulase induction. Results Here we show that CRT1 is a high-affinity cellobiose transporter, whose function can be complemented by the expression of other known cellobiose transporters. Expression of two sequence variants of the crt1 gene in Saccharomyces cerevisiae revealed that only the variant listed in the RUT-C30 genome annotation has the capability to transport cellobiose and lactose. When expressed in the $$\Delta$$ Δ crt1 strain, the variant listed in the QM6a genome annotation offers partial complementation of the cellulase induction, while the expression of the RUT-C30 variant or cellobiose transporters from two other fungal species fully restore the cellulase induction. Conclusions These results add to our knowledge about the fungal metabolism of cellulose-derived oligosaccharides, which have the capability of inducing the cellulase production in many species. They also help us to deepen our understanding of the T. reesei lactose metabolism, which can have important consequences as this sugar is used as the inducer of protein secretion in many industrial processes which employ this species.

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“…Although both ST2 and ST16 did not improve anaerobic utilization of XD compared with CDT-2, the presence of xylose or other simple sugars could be used to initiate the consumption of XD, according to our previous results [ 7 ]. Based on the rapid turn-off and turn-on of XD consumption in anaerobic conditions [ 7 ], we suspect that the XD transporters are internalized in the absence of hexose or xylose sugars [ 27 ]. Currently, how CDT-2 and ST16 are regulated in xylodextrin-only media remains unknown.…”

Section: Discussionmentioning

confidence: 99%

Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

Zhang

Acosta-Sampson

et al. 2017

PLoS ONE

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The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

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Internalization of Heterologous Sugar Transporters by Endogenous α-Arrestins in the Yeast Saccharomyces cerevisiae

Cited by 15 publications

References 54 publications

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillusoryzae

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillusoryzae

Studies on sugar transporter CRT1 reveal new characteristics that are critical for cellulase induction in Trichoderma reesei

Screening of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

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