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Exchange of water for sterol underlies sterol egress from a StARkin domain.

ABSTRACT: Previously we identified Lam/GramD1 proteins, a family of endoplasmic reticulum membrane proteins with sterol-binding StARkin domains that are implicated in intracellular sterol homeostasis. Here, we show how these proteins exchange sterol molecules with membranes. An aperture at one end of the StARkin domain enables sterol to enter/exit the binding pocket. Strikingly, the wall of the pocket is longitudinally fractured, exposing bound sterol to solvent. Large-scale atomistic molecular dynamics simulations reveal that sterol egress involves widening of the fracture, penetration of water into the cavity, and consequent destabilization of the bound sterol. The simulations identify polar residues along the fracture that are important for sterol release. Their replacement with alanine affects the ability of the StARkin domain to bind sterol, catalyze inter-vesicular sterol exchange and alleviate the nystatin-sensitivity of lam2Δ yeast cells. These data suggest an unprecedented, water-controlled mechanism of sterol discharge from a StARkin domain.

SUBMITTER: Khelashvili G

PROVIDER: S-EPMC6940019 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Exchange of water for sterol underlies sterol egress from a StARkin domain.

Khelashvili George G Chauhan Neha N Pandey Kalpana K Eliezer David D Menon Anant K AK

eLife 20191204

Previously we identified Lam/GramD1 proteins, a family of endoplasmic reticulum membrane proteins with sterol-binding StARkin domains that are implicated in intracellular sterol homeostasis. Here, we show how these proteins exchange sterol molecules with membranes. An aperture at one end of the StARkin domain enables sterol to enter/exit the binding pocket. Strikingly, the wall of the pocket is longitudinally fractured, exposing bound sterol to solvent. Large-scale atomistic molecular dynamics s ...[more]

PMID: 31799930

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Project description:Phytophthora species are devastating filamentous plant pathogens that belong to oomycetes, a group of microorganisms similar to fungi in morphology but phylogenetically distinct. They are sterol auxotrophic, but nevertheless exploit exogenous sterols for growth and development. However, as for now the mechanisms underlying sterol utilization in Phytophthora are unknown. In this study, we identified four genes in Phytophthora capsici that encode proteins containing a sterol-sensing domain (SSD), a protein domain of around 180 amino acids comprising five transmembrane segments and known to feature in sterol signaling in animals. Using a modified CRISPR/Cas9 system, we successfully knocked out the four genes named PcSCP1 to PcSCP4 (for P. capsici SSD-containing protein 1 to 4), either individually or sequentially, thereby creating single, double, triple, and quadruple knockout transformants. Results showed that knocking out just one of the four PcSCPs was not sufficient to block sterol signaling. However, the quadruple "all-four" PcSCPs knockout transformants no longer responded to sterol treatment in asexual reproduction, in contrast to wild-type P. capsici that produced zoospores under sterol treatment. Apparently, the four PcSCPs play a key role in sterol signaling in P. capsici with functional redundancy. Transcriptome analysis indicated that the expression of a subset of genes is regulated by exogenous sterols via PcSCPs. Further investigations showed that sterols could stimulate zoospore differentiation via PcSCPs by controlling actin-mediated membrane trafficking. Moreover, the pathogenicity of the "all-four" PcSCPs knockout transformants was significantly decreased and many pathogenicity related genes were downregulated, implying that PcSCPs also contribute to plant-pathogen interaction. IMPORTANCE Phytophthora is an important genus of oomycetes that comprises many destructive plant pathogens. Due to the incompleteness of the sterol synthesis pathway, Phytophthora spp. do not possess the ability to produce sterols. Therefore, these sterol auxotrophic oomycetes need to recruit sterols from the environment such as host plants to support growth and development, which seems crucial during pathogen-plant interactions. However, the mechanisms underlying sterol utilization by Phytophthora spp. remain largely unknown. Here, we show that a family of sterol-sensing domain-containing proteins (SCPs) consisting of four members in P. capsici plays a key role in sterol signaling with functional redundancy. Moreover, these SCPs play a role in different biological processes, including asexual reproduction and pathogenicity. Our study overall revealed the multiple functions of PcSCPs and addressed the question of how exogenous sterols regulate the development of heterothallic Phytophthora spp. via SSD-containing proteins.

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Exchange of water for sterol underlies sterol egress from a StARkin domain.

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Exchange of water for sterol underlies sterol egress from a StARkin domain.

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