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Article

Research article

J. Microbiol. Biotechnol. 2019; 29(1): 141-150

Published online January 28, 2019 https://doi.org/10.4014/jmb.1807.07050

Copyright © The Korean Society for Microbiology and Biotechnology.

A Role of YlBud8 in the Regulation of Cell Separation in the Yeast Yarrowia lipolytica

Li Yun Qing *, Xue Qing Jie , Yang Yuan Yuan , Wang Hui and Li Xiu Zhen

Department of Pathogenic Biology, Jining Medical University, Shandong 272067, China

Correspondence to:Li Yun Qing
liyunqing2013@whu.edu.cn

Received: July 24, 2018; Accepted: October 20, 2018

Abstract

The spatial landmark protein Bud8 plays a crucial role in bipolar budding in the budding yeast Saccharomyces cerevisiae. The unconventional yeast Yarrowia lipolytica can also bud in a bipolar pattern, but is evolutionarily distant from S. cerevisiae. It encodes the protein YALI0F12738p, which shares the highest amino acid sequence homology with S. cerevisiae Bud8, sharing a conserved transmembrane domain at the C-terminus. Therefore, we named it YlBud8. Deletion of YlBud8 in Y. lipolytica causes cellular separation defects, resulting in budded cells remaining linked with one another as cell chains or multiple buds from a single cell, which suggests that YlBud8 may play an important role in cell separation, which is distinct from the function of Bud8 in S. cerevisiae. We also show that the YlBud8-GFP fusion protein is located at the cell membrane and enriched in the bud cortex, which would be consistent with a role in the regulation of cell separation. The coiled-coil domain at the Nterminus of YlBud8 is important to the correct localization and function of YlBud8, as truncated proteins that do not contain the coiled-coil domain cannot rescue the defects observed in Ylbud8Δ. This finding suggests that a new signaling pathway controlled by YlBud8 via regulation of cell separation may exist in Y. lipolytica.

Keywords: YlBud8, cell polarity, cell separation, coiled-coil

Introduction

Cell polarization is central to the development of most organisms as it plays important roles in cell differentiation, division, cell-cell signaling, and cell migration [1]. Many cellular physiological activities are directional, including nutrient transport, nerve signal transmission, as well as cell movement, and cell polarity is necessary for the normal function of these and other cellular activities. Saccharomyces cerevisiae is a classic model organism. Its cells display significant polarity when sensing and responding to external or internal signals, and polar growth is involved in many steps of the cell cycle, including budding site selection, bud growth, mating, mycelial growth and cell separation [2, 3]. Cell separation is important for both increased cell number and cell differentiation, and is considered to be a special form of polar growth [4]. In budding yeast, both budding and cell separation require actin polarization and exocytosis. During the process of budding, polar growth is directed to the cortex of the bud to assemble a new bud, while at the later stage of the cell cycle, polar growth is directed to the bud neck to facilitate cytokinesis [5, 6]. Site selection in budding and cell division is nonrandom, but rather is controlled by spatial signals [7]. Thus, it is important to understand how spatial cues determine sites of polarized cell growth.

The molecular regulation of this process has been thoroughly studied in S. cerevisiae. A large number of spatial landmarks that are involved in polarized cell growth during bud site selection have been identified, which exist in and are expressed in different cell types. There are two sets of landmark cues that can mark new bud sites. In haploid cells, the polar sites are marked by the axial landmarks Axl1, Axl2, Bud3, and Bud4. In diploid cells, the polar sites are marked by the bipolar landmarks Rax1, Rax2, Bud8, and Bud9 [8, 9]. The bud-site GTPase Rsr1 and its regulators, the GTPase activating protein (GAP) Bud2 and the guanine nucleotide exchange factor (GEF) Bud5 make up the core module, which can recognize the positional cues that mark the poles of the haploid and diploid cells [10]. Then, the Rsr1 GTPase module can regulate the GTPase Cdc42, which associates with effector proteins to establish cell polarity, initiate polarized growth, and assemble the new bud at specific sites [11-13].

Spatial control of polar growth is not a phenomenon unique to S. cerevisiae, but exists ubiquitously in other yeasts. For example, the non-model yeast Yarrowia lipolytica buds in a bipolar pattern: mother cells can choose the bud site at either the proximal or distal poles to the preceding site of cytokinesis, whereas the daughter cells usually bud at the distal pole to the birth site, this process is thought to be controlled by spatial cues [14]. With Y. lipolytica, the bipolar bud takes on a very steady budding pattern that exists without the influence of cell type change, cell morphology change, or changes in the external environment; this characteristic is different from those of Candida albicans and S. cerevisiae [15, 16]. We are interested in the spatial landmarks that are involved in regulating the stable budding pattern of Y. lipolytica, a hemiascomycetous yeast species distantly related to the model organism S. cerevisiae. This species exhibits several special characteristics at the physiological, biochemical and metabolic levels and has recently become a model organism as unconventional yeast [17].

Research on the spatial landmark for bipolar budding in Y. lipolytica appears to be thus far absent. In S. cerevisiae, Bud8 and Bud9 are thought to act as bipolar spatial landmarks that provide spatial signals in diploid cells [18, 19]. Bud8 is a part of the distal landmark, which is required for distal bud site selection; Bud9 is localized to the proximal pole and is required for proximal pole selection [20]. These two proteins can interact with Bud5 to deliver the landmark signals to the Rsr1 GTPase module [9]. We had previously discovered that YlRsr1 is involved in the regulation of the bipolar budding pattern and cell separation in Y. lipolytica. Cells without YlRsr1 cannot separate from one another and form cell chains or multi- buds [21]. To understand whether spatial landmarks similar to Bud8 and Bud9 exist to provide spatial signals to the YlRsr1 GTPase module in Y. lipolytica, the amino acid sequences of Bud8 and Bud9 from S. cerevisiae were used in a BLAST search for homologue proteins in the NCBI database (https://www.ncbi.nlm.nih.gov/) where the optional organism is Y. lipolytica.

Here, we show that the Bud8 homologue YlBud8 plays a crucial role in cell separation, but is not involved in regulating bipolar budding. A YlBud8-GFP fusion protein can be located at the cell membrane and the small bud cortex, which is different from where S. cerevisiae Bud8 is located. We further show that the coiled-coil domain in YlBud8 is required for its normal function and localization.

Materials and Methods

Strains, Media and Growth Conditions

The Y. lipolytica strains used in this study are listed in Table 1. The construction of strains and strain source are described below or in Table 1. Escherichia coli strain DH5α was used for plasmids amplification. Y. lipolytica strains were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or in synthetic YNBD medium (0.67% yeast nitrogen base without amino acid, 2% glucose) at 30°C. YNBD media were supplemented with 20 mg/l uracil, 80 mg/l leucine, or both when required. Agar was added to 2% concentration for solid media, and glucose was used in place of glycerin to keep cells in their oval form in synthetic media.

Table 1 . Y. lipolytica strains used in this study..

StrainGenotypeSource
PO1aMATA leu2-270 ura3-30222
YLX403MATA leu2-270 ura3-302 Ylbud8Δ::loxR/PThis study
YLJ3MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 YlBUD8]This study
YLJ4MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 Ylbud8-1]This study
YLJ5MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 Ylbud8-2]This study
YLJ8MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-YlBUD8]This study
YLJ9MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-Ylbud8-1]This study
YLJ10MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-Ylbud8-2]This study
YLJ14MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 Ylbud8-3]This study
YLJ15MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 Ylbud8-4]This study
YLJ16MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-Ylbud8-3]This study
YLJ17MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-Ylbud8-4]This study


Construction of Plasmids and Yeast Strains

Plasmids and primers used in this study are respectively listed in Tables 2 and 3. YlBUD8 was deleted in the wild-type strain PO1a using homologous recombination, which was used in the deletion of YlTEC1 [23]. PCR was used to identify the mutants that carried the correct replacement of YlBud8. Plasmid pRRQ2 was transformed into the YlBUD8 deletion mutant to remove the selection marker YlURA3. We used PCR to confirm the deletion strains with the YlBUD8-5CK & YlBUD8-1R primers after the YlURA3 marker was removed.

Table 2 . Plasmids used in this study..

PlasmidDescriptionSource
pRRQ2CEN YlLEU2 hp4d-CREJean-Marc Nicaud
pWU24YlURA3 (178 bp promoter, ORF and 772 bp 3’ UTR) in pBlueScript KS(+)23
pWU24-YlBUD8YlBUD8 in pWU24This study
pWU25PYlBUD8 (680 bp promoter) in pWU24This study
pWU25-Ylbud8-1Ylbud8-1 in pWU25This study
pWU25-Ylbud8-2Ylbud8-2 in pWU25This study
pWU25-Ylbud8-3Ylbud8-3 in pWU25This study
pWU25-Ylbud8-4Ylbud8-4 in pWU25This study
pYL8loxR-YlURA3-loxP YlLEU2 in pBlueScript KS(+)23
pYL8-YlBUD8-PTPYlBUD8-loxR-YlURA3-loxP-TYlBUD8 in pYL8This study
pYL15PYlTEF1-EGFP in pINA44524
pYL15-YlBUD8PYlTEF1-EGFP-YlBUD8 in pYL15This study
pYL15-Ylbud8-1PYlTEF1-EGFP-Ylbud8-1 in pYL15This study
pYL15-Ylbud8-2PYlTEF1-EGFP-Ylbud8-2 in pYL15This study
pYL15-Ylbud8-3PYlTEF1-EGFP-Ylbud8-3 in pYL15This study
pYL15-Ylbud8-4PYlTEF1-EGFP-Ylbud8-4 in pYL15This study


Table 3 . Primers used in this study..

NameSequence (5’ → 3’)Use
YlBUD8-PFCGCGGATCCGAGATATAGCGTATGGTCAGCAmplification of PYlBUD8 and YlBUD8
YlBUD8-PRCCGGAATTCAGAAGTTGTAGTGAAGTAGCGAmplification of PYlBUD8
YlBUD8-TFACCCAAGCTTTGGTTGCTGGCGTTTTAACGAAmplification of TYlBUD8
YlBUD8-TRCCCAGCGTCGACTAGGAGTCTAGCCAAACGACCAmplification of TYlBUD8
YlBUD8-5CKACAACAGATGAAGAAGAGChecking Ylbud8Δ deletion
YlBUD8-1PFCGGGGTACCGACATGAAATCTCATGGCTTCAGAmplification of PYlBUD8
YlBUD8-1PRCCCATCGATAGAAGAAGTTGTAGTGAAGTAGAmplification of PYlBUD8
YlBUD8-1RACATGCATGCCGCTTTGTCAGACTGTCTACCChecking Ylbud8Δ deletion
YlBUD8-1FCCCATCGATTGCACGAACTCCACATTCCACCAmplification of YlBUD8
YlBUD8-2RCGCGGATCCAATGCTCAGTAATGACTGATGGAmplification of YlBUD8, Ylbud8-1, Ylbud8-2
YlBUD8-2FCCCATCGATATGCTCACCGACATTAACAACGAAmplification of Ylbud8-1
YlBUD8-3FCCCATCGATATGGTGGATAATATTGTCACAGAmplification of Ylbud8-2
YlBUD8-7FCCCATCGATATGAATGTCTCTGTTGACTCCTTAmplification of Ylbud8-3
YlBUD8-8FCCCATCGATATGGGCGAGGGTGAGGATGAGAmplification of Ylbud8-4
YlBUD8-4FTGCTCTAGAATGGACACGAAACGAAGCCTTTCAmplification of YlBUD8
YlBUD8-5FTGCTCTAGACTCACCGACATTAACAACGAGAmplification of Ylbud8-1
YlBUD8-6FTGCTCTAGAGTGGATAATATTGTCACAGTCAmplification of Ylbud8-2
YlBUD8-9FTGCTCTAGAAATGTCTCTGTTGACTCCTTAmplification of Ylbud8-3
YlBUD8-10FTGCTCTAGAGGCGAGGGTGAGGATGAGAmplification of Ylbud8-4


The plasmid pWU24-YlBUD8 was constructed to complement the Ylbud8Δ mutant. The 4,135-bp YlBUD8 gene containing promoter (845 bp) and a 282-bp 3’-untranslated region (UTR) was digested by ClaI and BamHI and then ligated into pWU24 (integrative, YlURA3) [24]. The plasmids were linearized by PmlI to introduce them into yeast cells for the complementation test.

To generate a version of pWU25 that tests the activity of truncated fragments of YlBud8, the 668-bp KpnI-ClaI YlBUD8 promoter was amplified by PCR and was inserted into pWU24. YlBud8 segments were inserted into the plasmid pWU25 digested by ClaI and BamHI, resulting in pWU25-Ylbud8-1, pWU25- Ylbud8-2, pWU25-Ylbud8-3, and pWU25-Ylbud8-4. The plasmids were also linearized, leading to Ylbud8Δ, which was used to identify the function of the four fragments.

The plasmid pYL15-YlBUD8 was constructed to examine the subcellular localization of YlBud8. To do this, the 3,280-bp YlBUD8 gene containing the full ORF and a 282-bp 3’-UTR sequence was amplified by PCR from genomic DNA and ligated into XbaI- and BamHI-digested pYL15 (CEN, YlLEU2, PYlTEF1-EGFP) [27], the N-terminus of YlBud8 was fused with EGFP. The truncated fragments were also amplified from genomic DNA and inserted into the XbaI- and BamHI-digested pYL15. The five plasmids were all introduced into Ylbud8Δ cells to examine the differences in localization of the different truncated fragments.

Yeast Transformation

The lithium acetate method was used to introduce plasmids to Y. lipolytica strains. This method has been used in the transformation of S. cerevisiae, but with Y. lipolytica, the cells were heat shocked at a lower temperature for a shorter period of time before plating on selective medium: 37°C for 15 min.

Western-Blot Analysis of Proteins

Cells of strain Ylbud8Δ carrying pYL15 or pYL15-YlBud8 segments were grown in YNBL+Ura medium at 30°C for 16 h. To extract total cellular proteins the Yeast Protein Extraction Reagent (Takara, Japan) was used. Proteins were separated by 8.0% SDS-PAGE. Mouse monoclonal antibody against GFP (Abcam, England) was used as primary antibody and horseradish peroxidase- conjugated goat anti-mouse IgG was used as secondary antibody.

Microscopy

Cell morphology was observed using an Olympus BX51 microscope (Japan) and a DP80 charge-coupled-device (CCD) camera. The images were acquired using CellSens Standard. For the count of the percentage of cells with abnormal morphology, a minimum of 200 cells were counted.

Results

Identification of YlBud8 in Y. lipolytica

In order to find the potential bipolar landmark in Y. lipolytica, the amino acid sequences of Bud8 and Bud9 from S. cerevisiae were used in a BLAST search and compared against the NCBI database (https://www.ncbi.nlm.nih.gov/) to find proteins that share the highest degree of similarity in amino acid sequences. The protein encoded by the ORF YALI0F12738 satisfied the above condition, sharing 11% identity with Bud8 and 10% identity with Bud9 from S. cerevisiae. Although these are not considered to be high degrees of homology, YALI0F12738 was the sequence sharing the highest degree of homology among Y. lipolytica proteins. We then analyzed the conserved domains of these three proteins use SMART (http://smart.embl-heidelberg.de/) and discovered that YALI0F12738p shares the same conserved transmembrane (TM) domain at its C-terminus and that this domain shares 23% and 22% amino acid sequence identity with the TM domains of Bud8 and Bud9, respectively (Fig. 1). In addition, we found that YALI0F12738 is 995 amino acids in length with a long N-terminus containing a coiled-coil domain. In comparison, Bud8 and Bud9 encode a 603-amino acid protein and a 547-amino acid protein, respectively, with a shorter N-terminus and without a coiled-coil domain, which may be the reason that the degree of homology between these proteins is so low. We named the Y. lipolytica homolog protein YlBud8 and analyzed its function.

Figure 1. Homology domains in Bud8 and Bud9. (A) Map of S. cerevisiae Bud8, Bud9 and Y. lipolytica YlBud8 domains, TM domain is shown in black and the coiled-coil domain is shown in gray. (B) Sequence alignment of the TM domains in S. cerevisiae Bud8, Bud9 and YlBud8. Black shading: identical residues; gray shading: similar residues.

YlBud8 Plays an Important Role in Cell Separation

S. cerevisiae cells lacking either Bud8 or Bud9 are normal in cell morphology and growth, with changes observed only in bipolar budding. To determine whether YlBud8 is required for bipolar budding, homologous recombination was used to delete YlBUD8 from the Y. lipilytica wild-type strain PO1a. We successfully obtained three mutants where YlBUD8 was correctly deleted, and the deletion was verified by PCR. When the wild-type genome was used as a template, we were able to obtain a PCR product of 5.1 kb, while a PCR product of 2.1 kb was obtained when the mutant genomes were used as the PCR template (Fig. 2), which indicated that YlBUD8 was correctly deleted. We then named the mutant YLX403 and observed its phenotype to help identify the function of YlBud8.

Figure 2. Verification of Y. lipolytica gene deletion mutant. (A) Schematic representation of YlBUD8 deletion procedure. The YlBUD8 ORF was first replaced by the loxR-YlURA3-loxP marker via homologous recombination. Then, the YlURA3 marker was removed. The location of the primers YlBud8-5CK & YlBud8-1R used for PCR is shown. (B) Verification of YlBUD8 deletion in three independent clones of the strain Ylbud8Δ::loxR/P. The primer pair YlBUD8-5CK/ YlBUD8-1R was used to amplify the YlBUD8 locus in strains PO1a (WT) and Ylbud8Δ::loxR/P. Expected amplified DNA fragments are 5.1 kb (WT) and 2.1 kb (Ylbud8Δ::loxR/P).

In YPD liquid medium, cells that are Ylbud8Δ exhibit bipolar budding, which is the same as observed in the wild-type PO1a (data not shown). This result suggests that YlBud8 is not involved in the regulation of bipolar budding in Y. lipolytica. In order to find the function of YlBud8, we examined Ylbud8Δ cells in yeast form and during filamentous growth. We did not observe a detectable growth defect in Ylbud8Δ cells at 30oC (data not shown), but cells showed morphological defects during yeast form and filamentous growth. In liquid YPD medium (yeast form), most Ylbud8Δ (80%, n = 200) cells remain linked to one another and form cell chains or multiple buds, while the wild-type PO1a forms single and oval form cells, and only 10% (n = 200) of the cells form cell chains (Figs. 3A and 3B). We also examined the cell morphology in YNBD and YNDC7 media that can promote mycelial growth to detect their ability to form pseudohyphae and hyphae. The PO1a strain formed an elongated morphology in YNBD medium and a few hyphae in YNDC7 medium. In YNBD medium, more pseudohyphae (83%, n = 200) could be observed in Ylbud8Δ cells than in wild-type cells (7%, n = 201) (Fig. 3C). The same phenomenon was observed when cells were grown in YNDC7 medium, which can induce hyphae growth, as the majority of cells (85%, n = 200) formed pseudohyphae where the length of a single cell is the same as that of the wild-type cell (Fig. 3D). When the intact plasmid pWU24- YlBUD8 was used for the complementation of the Ylbud8Δ strain, the cell morphology recovered well. This result proves that the defects we observed are caused by the deletion of YlBUD8 and that YlBUD8 was correctly deleted.

Figure 3. Phenotypes of Ylbud8Δ cells. (A) Cells of PO1a strain with plasmid pWU24 (WT/Vec) and YLX403 strain with plasmid pWU24 (Ylbud8Δ/Vec) or pWU24-YlBUD8 (Ylbud8Δ/ YlBUD8) were grown in liquid YPD medium at 30oC for 12 h, then were stained with Calcofluor white. (B) The percentage of single cells and cell chains were counted. (C and D) The same cells shown in A were grown in liquid YNBD and YNDC7 media at 30oC for 16 h. Scale bar, 5 μm.

Together, we conclude that YlBud8 is not involved in the regulation of bipolar budding but plays an important role in cell separation of Y. lipolytica during cell growth.

The Coiled-Coil Domain in YlBud8 is Important for Regulating Cell Separation

YlBud8 has a long N-terminus with a coiled-coil domain which is absent in Bud8 and Bud9 from S. cerevisiae. The coiled-coil domain is a common structural pattern that mediates protein-protein interactions [25]. Therefore, we wanted to find out whether the long N-terminus or the coiled-coil domain plays an important role in the normal function of YlBud8. We constructed four truncated constructs of YlBud8, named Ylbud8-1, Ylbud8-2, Ylbud8-3 and Ylbud8-4. The long N-terminus of YlBud8 was removed but the coiled-coil domain was retained in Ylbud8-1 and Ylbud8-3 while in Ylbud8-2 and Ylbud8-4 the long N-terminus and the coiled-coil domain were both removed (Fig. 4A). The truncated Ylbud8 segments were expressed in Ylbud8Δ cells under the control of the YlBud8 promoter to determine whether they can rescue the defects in cell morphology found in Ylbud8Δ. We examined the cell morphology of Ylbud8Δ cells expressing YlBud8 segments in YPD liquid. We found that YlBud8, Ylbud8-1, and Ylbud8-3 can rescue the cell morphology defect of Ylbud8Δ cells during yeast form growth (Fig. 4C). In YPD medium, 83% (n = 200) of Ylbud8Δ/Ylbud8-1 and 87% (n = 200) of Ylbud8Δ/Ylbud8-3 cells show normal cell shape and size. Only 17% or 13% of cells are connected with one another in short cell chains or multiple buds, which is close to the 8% (n = 221) short cell chains or multiple buds found in Ylbud8Δ/YlBud8 cells (Fig. 4B). These results suggest that the long N-terminus deletion in YlBud8 is largely functional in cell separation and further investigation will be needed to elucidate the function of the coiled-coil domain.

Figure 4. The coiled-coil domain in YlBud8 plays an important role in regulating cell separation. (A) Schematic representation of the domains of YlBud8 and the position of YlBud8 truncated segments Ylbud8-1, 2, 3, 4. (B) Cells of strain Ylbud8Δ integrated with PmlI-linearized plasmids pWU24-YlBud8 (YlBud8) and pWU25-Ylbud8 fragments were grown in liquid YPD medium for 12 h, and the percentage of single cells and cell chains was determined. (C) Same cells in B were stained with calcofluor white. Scale bar, 5 μm.

The truncated Ylbud8-2 and Ylbud8-4 segments were expressed in Ylbud8Δ cells under the control of the YlBUD8 promoter to study whether they can rescue the defect of Ylbud8Δ in cell morphology. Our results show that neither Ylbud8-2 nor Ylbud8-4 can rescue the cell morphology defect of Ylbud8Δ during yeast form growth. When grown in YPD medium, 75% (n = 200) of Ylbud8Δ/Ylbud8-2 and 79% (n = 200) of Ylbud8Δ/Ylbud8-4 cells are linked together with one another and form cell chains or multiple buds, which is similar to the phenotype of Ylbud8Δ mutants (Fig. 4B and 4C). Therefore, we conclude that the coiled- coil domain is essential for the function of YlBud8 to regulate cell separation, but the long N-terminus is not required for this purpose.

The Coiled-Coil Domain Plays an Important Role in the Localization of YlBud8

Protein localization is often linked to function. To obtain more information about the function of YlBud8, we fused EGFP to the N-terminus of YlBud8 to observe the location of EGFP-YlBud8. The fusion construct EGFP-YlBUD8 was expressed using the YlTEF1 promoter and was fully functional, as it rescued the cell morphology defect of Ylbud8Δ. The fusion protein can locate to the plasma membrane uniformly in unbudded cells, and is enriched in the bud cortex in both small-budded and large-budded cells (Fig. 5A). This result is consistent with the finding that YlBud8 may play an important role in cell separation.

Figure 5. The coiled-coil domain plays an important role in the localization of YlBud8. (A) Cells of strain YLX403 (Ylbud8Δ) carrying plasmids pYL15-YlBud8 fragments were grown in liquid YNBL medium supplemented with uracil at 30oC to observe GFP fluorescence. Scale bar, 5 μm. (B) Yeast strains in A were grown in liquid YNBL+Ura medium for 12 h and total proteins were extracted, which were separately subjected to 8.0% SDS-PAGE and immunoblotted with an anti-GFP antibody.

We also fused the truncated fragments of YlBud8 to EGFP at their N-terminus to study their localization. YlBud8 and the truncated fragments were all expressed correctly and stably as determined by immunoblotting with a monoclonal antibody against GFP (Fig. 5B). The functions of the fusion proteins are similar to the proteins that were expressed at the basal level in the pWU25 plasmid in the previous section. Fragments EGFP-Ylbud8-1 and EGFP-Ylbud8-3 localize to the same locations as EGFP- YlBud8, as they can be observed in the plasma membrane and the bud cortex. However, the fragments EGFP-Ylbud8-2 and EGFP-Ylbud8-4, which cannot rescue the cell separation defect of Ylbud8Δ, cannot locate to the cell membrane and bud cortex, but rather remain dispersed in the cytoplasm (Fig. 5A). The above results indicate that the localization of YlBud8 to the cell membrane and the bud cortex are essential to its function in cell separation, and that the mutants Ylbud8-2 and Ylbud8-4, which do not contain the coiled-coil domain, are incorrectly localized. This finding indicates that the coiled-coil domain is important to the localization of YlBud8, and that correct localization of YlBud8 may be a functional determinant.

Discussion

YlBud8 Plays Important Roles in Cell Separation

In S. cerevisiae, Bud8 and Bud9 are the essential components of the bipolar landmarks in diploid strains. The deletion of either Bud8 or Bud9 can change the bipolar budding pattern of the diploid strain, but has no influence on cell morphology and cell separation [26, 27]. We have identified YlBud8 in Y. lipolytica as a homolog of Bud8 and Bud9 and studied its function. The data we present above show that YlBud8 plays an important role in cell separation, as YlBud8 deletion mutants cannot separate from one another and ultimately form cell chains or multi-bud cells. The program TMHMM server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0) predicts that there are two short TM domains at the C-terminus of YlBud8 and we speculate that the N-terminus of YlBud8 may be located to the extracytoplasmic space, which can provide a spatial basis for YlBud8 to interact with other proteins that can regulate cell separation. In Y. lipolytica, deletion of YlRsr1 can also result in the formation of cell chains or multi-bud cells, which is similar to the phenotypes observed with YlBud8 deletion cells [21]. We speculate that YlBud8 may act as the spatial landmark for cell separation and transmit spatial signals to YlRsr1, so that the function of YlBud8 in cell separation may be mediated by the Bud8 →Rsr1 →Cdc24 → Cdc42 signaling pathway.

We propose two reasons for the differences between the functions of the two homologous proteins Bud8 and YlBud8. Firstly, the structure of YlBud8 differs significantly from that of Bud8 as it has a long N-terminus and a coiled- coil domain at the N-terminus which is non-existent in S. cerevisiae Bud8. Coiled-coils are known as domains that can mediate protein-protein interactions [25]. We speculate that the coiled-coil domain endows YlBud8 with novel functions that may influence the roles it plays in Y. lipolytica. cr proteins through the coiled-coil domain to take part in cell separation, but not in bipolar budding. Secondly, the location of YlBud8 differs from that of Bud8 in S. cerevisae. In S. cerevisiae, Bud8 can localize to the bud tips and presumptive bud sites, which provides the spatial basis for the role played by Bud8 in the regulation of bipolar budding [26]. However, in Y. lipolytica, YlBud8 localizes to the cell membrane and the small bud cortex, but cannot be observed at the bud neck or the presumptive bud sites. This localization is consistent with the data indicating that YlBud8 is not involved in the regulation of bipolar budding. Besides, Bud8 homolog proteins from other species also have different functions in different biological processes. For example, in Cryptococcus neoformans, BUD8 was significantly up-regulated in the H99 strain, which demonstrates higher virulence than wild-type strains, but its function in cell morphology requires further investigation [28].

The Coiled-Coil Domain Is Essential for the Function of YlBud8

The coiled-coil domain is an abundant structural motif that is present in approximately 10% of all proteins of any given species [29]. The amino acids in this domain can interact among themselves to form amphiphilic α-helices, which then form a super-helix, i.e. the coiled-coil [30]. Coiled-coil domains have been visualized as rod-like spacers separating functional domains and they always act as protein scaffolds to mediate interactions between proteins [31, 32]. A coiled-coil domain exists at the N- terminus of YlBud8 and we hypothesize that this domain can mediate the interaction between YlBud8 and other proteins to enable YlBud8 to perform its normal functions. To test this, truncated fragments of YlBud8 were constructed. Our results show that the fragments Ylbud8-1 and Ylbud8-3, which contain the coiled-coil domain but not the long N-terminus of YlBud8, can localize to the correct cellular sites, such as the cell membrane and the small bud cortex and perform their normal functions, as they are able to complement the cellular defects observed in the Ylbud8Δ mutant. However, when the coiled-coil domain was removed, as with Ylbud8-2 and Ylbud8-4 fragments, the fragments were neither able to perform normal functions nor localize to normal sites. These results indicate that it is the coiled-coil domain, but not the long N-terminus that plays important roles in the normal function of YlBud8. However, whether downstream and upstream signaling proteins of YlBud8 can act on YlBud8 through the coiled- coil domain has not been confirmed and further investigations will be required.

To date, no other homologs of Bud8 from organisms other than S. cerevisiae and C. neoformans have been reported, and our study will enrich the known functional diversity of Bud8 homologs. There are still many questions at present on the function of these proteins and further tests are required to determine if YlBud8 can act on YlRsr1 to play effector roles in cell separation. The functions of other domains of YlBud8 also require further study.

Acknowledgments

We thank X-D Gao for his support, helpful discussions and providing plasmids and yeast strains. This work was supported by the National Natural Science Foundation of China (no. 31500056), Scientific Research Fund for Ph.D. of Jining Medical University (no. JY2015BS13) and Supporting Fund for Teachers’ Research of Jining Medical University (no. JY2017KJ012).

Conflict of Interest


The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Homology domains in Bud8 and Bud9. (A) Map of S. cerevisiae Bud8, Bud9 and Y. lipolytica YlBud8 domains, TM domain is shown in black and the coiled-coil domain is shown in gray. (B) Sequence alignment of the TM domains in S. cerevisiae Bud8, Bud9 and YlBud8. Black shading: identical residues; gray shading: similar residues.
Journal of Microbiology and Biotechnology 2019; 29: 141-150https://doi.org/10.4014/jmb.1807.07050

Fig 2.

Figure 2.Verification of Y. lipolytica gene deletion mutant. (A) Schematic representation of YlBUD8 deletion procedure. The YlBUD8 ORF was first replaced by the loxR-YlURA3-loxP marker via homologous recombination. Then, the YlURA3 marker was removed. The location of the primers YlBud8-5CK & YlBud8-1R used for PCR is shown. (B) Verification of YlBUD8 deletion in three independent clones of the strain Ylbud8Δ::loxR/P. The primer pair YlBUD8-5CK/ YlBUD8-1R was used to amplify the YlBUD8 locus in strains PO1a (WT) and Ylbud8Δ::loxR/P. Expected amplified DNA fragments are 5.1 kb (WT) and 2.1 kb (Ylbud8Δ::loxR/P).
Journal of Microbiology and Biotechnology 2019; 29: 141-150https://doi.org/10.4014/jmb.1807.07050

Fig 3.

Figure 3.Phenotypes of Ylbud8Δ cells. (A) Cells of PO1a strain with plasmid pWU24 (WT/Vec) and YLX403 strain with plasmid pWU24 (Ylbud8Δ/Vec) or pWU24-YlBUD8 (Ylbud8Δ/ YlBUD8) were grown in liquid YPD medium at 30oC for 12 h, then were stained with Calcofluor white. (B) The percentage of single cells and cell chains were counted. (C and D) The same cells shown in A were grown in liquid YNBD and YNDC7 media at 30oC for 16 h. Scale bar, 5 μm.
Journal of Microbiology and Biotechnology 2019; 29: 141-150https://doi.org/10.4014/jmb.1807.07050

Fig 4.

Figure 4.The coiled-coil domain in YlBud8 plays an important role in regulating cell separation. (A) Schematic representation of the domains of YlBud8 and the position of YlBud8 truncated segments Ylbud8-1, 2, 3, 4. (B) Cells of strain Ylbud8Δ integrated with PmlI-linearized plasmids pWU24-YlBud8 (YlBud8) and pWU25-Ylbud8 fragments were grown in liquid YPD medium for 12 h, and the percentage of single cells and cell chains was determined. (C) Same cells in B were stained with calcofluor white. Scale bar, 5 μm.
Journal of Microbiology and Biotechnology 2019; 29: 141-150https://doi.org/10.4014/jmb.1807.07050

Fig 5.

Figure 5.The coiled-coil domain plays an important role in the localization of YlBud8. (A) Cells of strain YLX403 (Ylbud8Δ) carrying plasmids pYL15-YlBud8 fragments were grown in liquid YNBL medium supplemented with uracil at 30oC to observe GFP fluorescence. Scale bar, 5 μm. (B) Yeast strains in A were grown in liquid YNBL+Ura medium for 12 h and total proteins were extracted, which were separately subjected to 8.0% SDS-PAGE and immunoblotted with an anti-GFP antibody.
Journal of Microbiology and Biotechnology 2019; 29: 141-150https://doi.org/10.4014/jmb.1807.07050

Table 1 . Y. lipolytica strains used in this study..

StrainGenotypeSource
PO1aMATA leu2-270 ura3-30222
YLX403MATA leu2-270 ura3-302 Ylbud8Δ::loxR/PThis study
YLJ3MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 YlBUD8]This study
YLJ4MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 Ylbud8-1]This study
YLJ5MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 Ylbud8-2]This study
YLJ8MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-YlBUD8]This study
YLJ9MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-Ylbud8-1]This study
YLJ10MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-Ylbud8-2]This study
YLJ14MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 Ylbud8-3]This study
YLJ15MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [Integrative URA3 Ylbud8-4]This study
YLJ16MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-Ylbud8-3]This study
YLJ17MATA leu2-270 ura3-302 Ylbud8Δ::loxR/P [CEN LEU2 EGFP-Ylbud8-4]This study

Table 2 . Plasmids used in this study..

PlasmidDescriptionSource
pRRQ2CEN YlLEU2 hp4d-CREJean-Marc Nicaud
pWU24YlURA3 (178 bp promoter, ORF and 772 bp 3’ UTR) in pBlueScript KS(+)23
pWU24-YlBUD8YlBUD8 in pWU24This study
pWU25PYlBUD8 (680 bp promoter) in pWU24This study
pWU25-Ylbud8-1Ylbud8-1 in pWU25This study
pWU25-Ylbud8-2Ylbud8-2 in pWU25This study
pWU25-Ylbud8-3Ylbud8-3 in pWU25This study
pWU25-Ylbud8-4Ylbud8-4 in pWU25This study
pYL8loxR-YlURA3-loxP YlLEU2 in pBlueScript KS(+)23
pYL8-YlBUD8-PTPYlBUD8-loxR-YlURA3-loxP-TYlBUD8 in pYL8This study
pYL15PYlTEF1-EGFP in pINA44524
pYL15-YlBUD8PYlTEF1-EGFP-YlBUD8 in pYL15This study
pYL15-Ylbud8-1PYlTEF1-EGFP-Ylbud8-1 in pYL15This study
pYL15-Ylbud8-2PYlTEF1-EGFP-Ylbud8-2 in pYL15This study
pYL15-Ylbud8-3PYlTEF1-EGFP-Ylbud8-3 in pYL15This study
pYL15-Ylbud8-4PYlTEF1-EGFP-Ylbud8-4 in pYL15This study

Table 3 . Primers used in this study..

NameSequence (5’ → 3’)Use
YlBUD8-PFCGCGGATCCGAGATATAGCGTATGGTCAGCAmplification of PYlBUD8 and YlBUD8
YlBUD8-PRCCGGAATTCAGAAGTTGTAGTGAAGTAGCGAmplification of PYlBUD8
YlBUD8-TFACCCAAGCTTTGGTTGCTGGCGTTTTAACGAAmplification of TYlBUD8
YlBUD8-TRCCCAGCGTCGACTAGGAGTCTAGCCAAACGACCAmplification of TYlBUD8
YlBUD8-5CKACAACAGATGAAGAAGAGChecking Ylbud8Δ deletion
YlBUD8-1PFCGGGGTACCGACATGAAATCTCATGGCTTCAGAmplification of PYlBUD8
YlBUD8-1PRCCCATCGATAGAAGAAGTTGTAGTGAAGTAGAmplification of PYlBUD8
YlBUD8-1RACATGCATGCCGCTTTGTCAGACTGTCTACCChecking Ylbud8Δ deletion
YlBUD8-1FCCCATCGATTGCACGAACTCCACATTCCACCAmplification of YlBUD8
YlBUD8-2RCGCGGATCCAATGCTCAGTAATGACTGATGGAmplification of YlBUD8, Ylbud8-1, Ylbud8-2
YlBUD8-2FCCCATCGATATGCTCACCGACATTAACAACGAAmplification of Ylbud8-1
YlBUD8-3FCCCATCGATATGGTGGATAATATTGTCACAGAmplification of Ylbud8-2
YlBUD8-7FCCCATCGATATGAATGTCTCTGTTGACTCCTTAmplification of Ylbud8-3
YlBUD8-8FCCCATCGATATGGGCGAGGGTGAGGATGAGAmplification of Ylbud8-4
YlBUD8-4FTGCTCTAGAATGGACACGAAACGAAGCCTTTCAmplification of YlBUD8
YlBUD8-5FTGCTCTAGACTCACCGACATTAACAACGAGAmplification of Ylbud8-1
YlBUD8-6FTGCTCTAGAGTGGATAATATTGTCACAGTCAmplification of Ylbud8-2
YlBUD8-9FTGCTCTAGAAATGTCTCTGTTGACTCCTTAmplification of Ylbud8-3
YlBUD8-10FTGCTCTAGAGGCGAGGGTGAGGATGAGAmplification of Ylbud8-4

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