Identification and Characterization of the Vesicle Protein YlSec15 in <i>Yarrowia lipolytica</i>

doi:10.13523/j.cb.20190305

China Biotechnology

2019, Vol. 39

Issue (3): 29-36 DOI: 10.13523/j.cb.20190305

Identification and Characterization of the Vesicle Protein YlSec15 in Yarrowia lipolytica

Kai-li CHEN¹,Fu-tao ZHANG¹,Dong-yue WANG¹,Qian ZHANG¹,Yun-qing LI^2,^**(

)

1 School of Clinical Medicine, Jining Medical University, Jining 272067,China
2 Department of Pathogenic Biology, Jining Medical University, Jining 272067, China

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Abstract

Yarrowia lipolytica is a budding yeast and the budding position which determined the division plane is not selected randomly but determined by the distance of the last division site, that is, bud in the bipolar budding pattern. At present, the molecular regulation pathway of the bipolar budding is not well studied. In this report, the characterization of the vesicle protein YlSec15 was studied through the localization and overexpression. YlSec15 can locate inside the small buds and the neck of the middle or big buds, indicating the patterns of polar localization. Overexpression of YlSec15 in the yeast can inhibit the formation of hyphae and change budding pattern of cells, from bipolar to random. The causes of these changes may be due to the delocalization to the polar site of the excessive YlSec15. Besides, the normal localization of YlSec15 dependent on Ylras2. Based on this result, we speculate that YlSec15 may be involved in regulating the polar growth of cells through the signal pathway which is mediated by YlRas2. The discovery in this paper will enrich the molecular regulation mechanism of the bipolar budding and confirm the relationship between polar growth and vesicle transport in Yarrowia lipolytica.

Key words： Yarrowia lipolytica Bipolar budding Hyphae Vesicle protein YlSec15

Received: 27 September 2018 Published: 12 April 2019

ZTFLH:

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Corresponding Authors: Yun-qing LI E-mail: liyunqing2013@whu.edu.cn

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	Kai-li CHEN
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	Yun-qing LI

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Kai-li CHEN,Fu-tao ZHANG,Dong-yue WANG,Qian ZHANG,Yun-qing LI. Identification and Characterization of the Vesicle Protein YlSec15 in Yarrowia lipolytica. China Biotechnology, 2019, 39(3): 29-36.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190305 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I3/29

菌株	基因型	来源
PO1a	MATA leu2-270 ura3-302	13
YLX260	MATA leu2-270 ura3-302 Ylrsr1∷loxR/P	8
YLX81	MATA leu2-270 ura3-302 Ylras2	9
YLJ4	MATA leu2-270 ura3-302 [Integrative URA3 YlSec15↑]	This study
YLJ8	MATA leu2-270 ura3-302 [CEN LEU2 YlSEC15-EGFP]	This study
YLJ9	MATA leu2-270 ura3-302 [CEN LEU2 EGFP-YlSec15↑]	This study
DH5	F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15Δ (lacZYA-argF) U169, hsdR17 ( $r K ?$ $m K +$ ), λ-	Takara, Japan

Table 1 Strains used in this study

Table 2 Primers used in this study

Fig.1 Isolation and identification of YlSec15 (a) Schematic representation of the domain structure of C. albicans CaSec15, S. cerevisiae ScSec15 and Y. lipolytica YlSec15. The conserved Sec15 superfamily domain is marked in gray (b) Amino acid sequence alignment of Sec15 superfamily domain in CaSec15, ScSec15, and YlSec15. Identical and similar residues are highlighted in black and gray, respectively

Fig.2 Localization of YlSec15-EGFP in cells Cells of wild type strain PO1a carrying plasmid pYL14-YlSec15 were grown in solid YNBD +Ura medium at 30℃ for 16 hours, then GFP fluorescence was observed under the oil microscope. Bar:5μm

Fig.3 YlSec15 is involved in regulating cell morphology (a) Cells of strain PO1a with plasmids pYL4 or pYL4-YlSec15 were grown in YPD solid medium at 30℃ for 3 days, then the colonial morphology was examined (b) Edge of the colonies in (a) was examined by a low-power microscope with 10×10 (c) Cells as in (a) were incubated in liquid YPD and YNDC7 mediums for 12 hours and 16 hours, respectively. Bar:5μm

Fig.4 Overexpression of YlSec15 influence the bipolar budding of cells (a) Cells of wild type strain PO1a with plasmids pYL4 or pYL4-YlSec15 were grown in solid YPD medium at 30℃ for 12 hours, then stained with Calcofluor white to identify patterns of bud-site selection (b) Budding patterns of cells in (a), the percentage of bipolar and random budding cells was counted. Bar:5μm

Fig.5 Distribution of excess YlSec15 in cells Cells of wild type strain PO1a with plasmid pYL15-YlSec15 were grown in solid YNBD+Ura medium at 30℃ for 16 hours, then GFP fluorescence was observed. Bar: 5μm

Fig.6 Localization of YlSec15-EGFP in Ylrsr1Δ and Ylras2Δ strains Cells of strains Ylrsr1Δ or Ylras2Δ with plasmid pYL14-YlSec15 were grown in solid YNBD+Ura medium at 30℃ for 16 hours, then GFP fluorescence was observed. Bar: 5μm


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