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The Metarhizium anisopliae Perilipin Homolog MPL1 Regulates Lipid…

- - - - 2.4-fold fewer LDs than WT
      - WT, the Mpl1 germ tubes are thinner
        and apparently analogous to the “lean” phenotype of perilipindeficient mice
      - 50% o fMpl1 germ tubes lack visible LDs.
    - - During germination of WT conidia in minimal medium, thetotal number of LDs diminishes by 62% indicative of lipid degradation, and the remaining LDs migrate into the germ tube apexes
    - - formation of appressoria on locust hind wings
        
        Mpl1 appressoria frequently contained no
        LDs at all
        
        After breaching the insect cuticle, Metarhizium produces
        variably shaped hyphal bodies for dispersal in the hemocoele
        
        throughout hyphal body cells,
        
        the Mpl1 mutants have fewer
    - - more dramatic difference in lipid levels ( 7-fold) was observed when germinating the WT and Mpl1 conidia in a minimal medium
        
        Mpl1 activity might adjust storage of lipids when nutrients are available to ensure
        extended survival when food supply is limiting
        
        8% increase in lipid content in conidia harvested fromPDAplus
        OA as compared with PDA alone was not statistically significantly
    - - No
        significant differences in lipid content
        
        noninductive media (YPD and 2% raffinose) or inductive medium (2% galactose) with or without the addition of OA.
        
        yeast cells that had not produced MPL1 rapidly
        reduced their lipid content during starvation
    - - Cap20” transcript of the Mpl1 homolog in the
        plant pathogen C. gloeosporioides is present in conidia
      - the role of Mpl1 on appressorium formation/function in M. anisopliae, we induced appressorial differentiation on locust hind wings
        
        occurs after nuclear division with a septum being formed between the appressorium and conidial mother cell
        
        only 10% of the mutant
        germlings
    - - serial concentrations of PEG-8000 shows that the loss of Mpl1
        results in a dramatic reduction of turgor pressure
        
        lipolysis of LDs and consequent production of
        solutes produce turgor in the wild type
        
        significant reduction of
        mortality and speed of kill by Mpl1 as compared with the WT.
        
        mutant failed to achieve 50% mortality before the insects pupated
        
        when the cuticle was bypassed by injecting spores directly into the hemocoele, the speed of kill by the WT (LT50 4.42 days) and the mutant (LT50 4.72 days) were not significantly different (p 1.58, t 0.13)
        
        1 more item...
  - - - lipid targeting
        functions reside
        
        full open reading frame of Mpl1 encodes a protein of 183 amino acids
        
        N terminus of Per A (17, 18), the MPL1 protein contains N-terminal -strands, three moderately hydrophobic regions (H1, H2, and H3), and an acidic region (129–136 in MPL1) before H2.
        
        like Per A, MPL1 has multiple phosphorylation sites, including a consensus cAMP-dependent
        protein kinase phosphorylation site at position 96 in a region conserved with Per A
    - - expressed during appressorial formation by the plant pathogen C. gloeosporioides and critical for virulence
        
        but showed no similarities between both of them using blast
    - - Each pezizomycotinal fungus has only a single perilipinlike
        gene in contrast to animals encoding three perilipin genes (A, B,and C)
    - - strong expression of Mpl1 when the fungus was grown in nutrient-rich media (insect hemolymph or SDB) as compared with nutrient-poor media
        
        The addition of fatty acids to mammalian cells stimulates lipid accumulation and increases intracellular levels of perilipin
        
        transcription of Mpl1 by M. anisopliae germlings was up-regulated within 30 min following the addition of oleic acid to minimal medium (MM)
      - Conidia contained many large LDs and demonstrated a strong enrichment of Mpl1 transcripts.
        
        Transcription levels decreased during germination
        
        And LD numbers dropped by 40% between 6 and 12 h as lipid stores are mobilized during germ tube elongation
        
        Results suggest that Mpl1 regulation parallels lipid storage.
    - - determined whether LD localization is a general quality of MPL1 by expressing it with the Gal1-inducible promoter in the yeast S. cerevisiae, which is a system that lacks endogenous perilipin-like proteins.
        
        Consistent with GFP fusions of mammalian PAT proteins, i.e. perilipin A, TIP47, and adipocyte differentiation-related protein, N-terminal fusion of MPL1 with GFP did not disrupt the ability of the protein to localize to lipid vesicles.
        
        Transformed yeast and Metarhizium cells treated with the neutral lipid stain NR colocalized with the GFP signal confirming that MPL1 is binding to LDs
        
        No additional diffuse cytoplasmic signal was seen with either GFP or NR
        
        The expression patterns and the intracellular localization of MPL1 are therefore consistent with the proposal that the protein plays a regulatory role in global triacylglycerol (TAG) storage by acting at the level of LDs.
- - - - perilipin forms
        a barrier that restricts the access of cytosolic lipases
      - During food deprivation, perilipin is phosphorylated by protein kinase A
        
        barrier function of perilipin is attenuated, and lipolysis increases.
    - - human perilipin variants are also
        associated with obesity or leanness
  - - - produces a single mammalian perilipin homolog we
        designated as Mpl1 for Metarhizium perilipin-like protein
- - - - Target and anchor Per A to LDs
      - Target and anchor Per A to LDs
  - - - Germ tubes of ^Mpl1 are slimmer than those of the wild type Metarhizium : similar to the lean Per A knock-out mouse under diet control
        
        Perilipin-like proteins in the most diverse eukaryotes share ancestrak function
  - - - But the yeast genome does not have a perilipin homolog
      - Mpl1 were unable to make use of their stored lipids during starvation
        
        Yeast has lost or never acquired phosphorylation mechanism for removing perilipin-like proteins to access LDs
  - - - Regulate the accumulation of glycerol, cell compartmentalization required to generate appressorial tugor
    - - Plant pathogen C. gloeosporioides also have diminished virulence
  - - - Mpl1 is up-regulated (2-fold) in aging fungal sectors
      - Degenerated fungus demonstrates decreased levels of and variable rates of lipid metabolism
    - - Identify the components and mechanisms of energy homeostasis and how that relates to other physiological process including aging