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Fungal SPFH Proteins: Comparison
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Please note this is a comparison between Version 2 by Catherine Yang and Version 4 by Catherine Yang.

Integral membrane proteins from the ancient SPFH (stomatin, prohibitin, flotillin, HflK/HflC) protein superfamily are found in nearly all living organisms. Mammalian SPFH proteins are primarily associated with mitochondrial functions but also coordinate key processes such as ion transport, signaling, and mechanosensation. In addition, SPFH proteins are required for virulence in parasites.

  • SPFH
  • prohibitin
  • stomatin
  • mitochondria
  • fungi

1. Introduction

The SPFH protein family is present in all domains of life. Proteins of this family are characterized by a conserved SPFH domain and diverge highly at their N- and C-terminal regions [1][2][3][4]. Furthermore, the distribution of SPFH proteins across species varies [1][2][4]. Proteomic and cellular analyses identified SPFH proteins in various cellular membranes, such as the inner mitochondrial membrane and plasma membrane [5][6][7][8][9][10][11][12]. SPFH proteins also localize to the endoplasmic reticulum and lysosome/vacuole [5][13][14]. SPFH protein overproduction in mammals, nematodes, yeast, and mice causes a broad array of phenotypes, including drug resistance, aging, apoptosis, and tumorigenesis [15][16][17]. Biochemical events dependent on SPFH proteins include palmitoylation and oligomerization and have supported a hypothesis that SPFH proteins are membrane scaffolds [18]. In vitro biochemical results showed that human stomatin protein binds directly to cholesterol and actin mainly through key amino acid sequences in the C-terminus [19]. Moreover, sequences in the SPFH domain are required for SPFH protein homo-oligomerization [19][20]. However, the details underlying the molecular function of SPFH proteins are limited.

2. SPFH Protein Function in Non-Pathogenic Fungi

In the baker’s yeast Saccharomyces cerevisiae, the sole SPFH proteins, prohibitin 1 and prohibitin 2 (Phb1, Phb2), form ring-shaped complexes within the inner mitochondrial membrane and are associated with several mitochondrial functions [9]. Phb1 and Phb2 interact with Mdm33 to regulate mitochondrial ultrastructure and shape [21]. In addition, Phb1 and Phb2 interact with the chaperones Atp10 and Atp23 to assist formation of F1F0-ATP synthase [22]. Depletion of prohibitins reduces yeast life span and is characterized by abnormal mitochondrial structure and delayed mitochondrial segregation to budding daughter cells [23][24][25].

Synthetic genetic arrays using a phb1Δ mutant strain identified 35 genes that are required for viability or normal growth [23]. Interestingly, 31 of these genes encode mitochondrial proteins. 19 of these genes were associated with respiratory chain assembly and maintenance of mitochondrial structure. Major PHB1 genetic partners include YTA10, YTA11, and YME1 [23]. These genes encode proteins which belong to the conserved, ATP-dependent mitochondrial m-AAA protease family, which maintain the mitochondrial proteome [26]. Other PHB1 genetic partners include the cytochrome c complex subunit-encoding genes, COX6 and COX24 [23]. In addition, 8 genes are required for the synthesis of the mitochondrial membrane lipids, cardiolipin and phosphatidylethanolamine. These partners include the highly conserved genes, UPS1 and UPS2 [23]. Lastly, prohibitin function and localization was associated with the presence of the yeast [PSI+] prion. Proteomic analysis revealed that aberrant mitochondrial function observed in [PSI+] prion yeast strains was caused, in part, by Phb1 mislocalization in the cytoplasm [27]. See Figure 1 for a summary of SPFH function in S. cerevisiae.

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Figure 1. SPFH protein distribution and functions in S. cerevisiae. Phb1 and Phb2 form ring-shaped complexes that localize to the inner mitochondrial membrane. Known mitochondrial functions associated with the Phb1/Phb2 complex are depicted in the blue boxes. Figure adapted from references [9][21][27][23][22][24] and created with Biorender.com.

SPFH function has also been characterized in other non-pathogenic fungi. In the fission yeast Schizosaccharomyces pombe, Phb1 and Phb2 localize to the mitochondria [28]. Overexpression or deletion of the phb2 gene caused resistance to various antifungal drugs including terbinafine, fluconazole, amphotericin B, and clotrimazole [28]. Moreover, increased production of intracellular nitric oxide and reactive oxygen species were observed in Phb2 overexpression or deletion strains [28]. In contrast, only a Δphb1 deletion strain was resistant to antifungal drugs [28]. Additional genetic evidence showed that mitochondrial dysfunction caused by phb2 deletion and overexpression activated the oxidative stress response transcriptional regulator, Pap1, thus linking prohibitins to stress response signaling [28]. Paradoxically, S. cerevisiae phb2Δ mutants were sensitive to fluconazole, amphotericin B, and clotrimazole, highlighting the differences of SPFH protein function in different yeast species [28]. However, the basis of this phenotype is unknown.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS ) analyses on mitochondrial extracts identified three SPFH proteins (Phb1, Phb2 and Slp2) in the filamentous fungus, Neurospora crassa [29]. Consistent with the structural dynamics of prohibitins from mammals, nematodes, and yeast, N. crassa Phb1 and Phb2 localized to the inner mitochondrial membrane and formed large membrane complexes of various sizes. Notably, one high molecular weight prohibitin complex co-migrated with m-AAA protease MAP-1. This suggests that m-AAA proteins may physically interact with prohibitins in N. crassa, similar to observations with S. cerevisiae prohibitins [29]. Moreover, the stomatin, Slp2, was found to co-migrate in a high molecular weight complex with the N. crassa i -MMM protease homolog, IAP-1, suggesting that Slp2 and IAP-1 physically interact in the inner mitochondrial membrane [29]. Taken together, these findings demonstrate the importance of SPFH protein function in mitochondrial ultrastructure, respiratory function, and antifungal drug resistance.

3. SPFH Protein Function in Pathogenic Fungi

In fungal pathogens, mitochondria are required for virulence determinants including morphogenesis, drug susceptibility, cell wall biogenesis, and biofilm formation [30][31][32]. The knowledge underlying the molecular and cellular aspects of mitochondrial function in human pathogenic fungi is based primarily on studies in the opportunistic pathogen, Candida albicans. Mitochondrial function is critical for C. albicans commensalism and virulence [30]. Indeed, C. albicans cells treated with respiratory inhibitors display aberrant cell wall structure and increased macrophage recognition [33]. Moreover, mutations to fungal-specific mitochondrial genes, such as GOA1, NUO3, NUO4, and GEM1, attenuate virulence [34][35][36][37]. Genome-wide transcriptional profiling revealed that genes encoding proteins with mitochondrial functions are significantly upregulated following cell wall damage or osmotic stress [38][39][40]. 

The C. albicans genome includes five SPFH family members: PHB1, PHB2, PHB12, SLP2, and SLP3 (stomatin-like protein 3) [41]. Recent studies have shown that SLP3 transcription and protein localization significantly increases following treatment with oxidative, osmotic, cell wall, or plasma membrane stress agents, categorizing SLP3 as a general stress response gene [5][39][40]. Fluorescence imaging revealed that Slp3p forms visible puncta along the plasma membrane similar to mammalian stomatin complexes [5][42]. Slp3 plasma membrane localization was also confirmed via liquid chromatography-mass spectrometry (LC-MS/MS) and MALDI-TOF analysis on C. albicans plasma membrane extracts [6]. Deletion of Slp3p has no effect on growth under nutrient-rich conditions, environmental stress conditions, or antifungal drug treatment [5]. Moreover, the absence of Slp3p does not result in any apparent cell structure abnormality, organelle malfunction, or ion transport defects [5]. Overexpression of Slp3, however, does result in hypersensitivity to reactive oxygen species and mitochondrial depolarization specifically in yeast-phase cells [5].

C. albicans Slp2, Phb1, Phb2, and Phb12 each contain a putative mitochondrial localization signal motif (the reseaourchers' preliminary findings). We observed Slp2 mitochondrial localization (the researchers'our preliminary findings), suggesting that the mitochondrial functions for prohibitins and Slp2 may be conserved. See Figure 2 for a summary of C. albicans SPFH protein function.

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Figure 2. SPFH protein distribution and function in C. albicans. Illustration depicting the cellular localization of SPFH family members Slp2, Slp3, Phb1, Phb2, and Phb12. Asterisks (*) beside Phb1, Phb2, and Phb12 denote that cellular localization of these proteins are only predicted and have not been experimentally confirmed. Biochemical and cytological analyses demonstrated that Slp3 localizes to the plasma membrane and vacuole, and Slp2 localizes to the mitochondria. Known functions associated with Slp3 are depicted in the blue boxes. Slp3 production is increased in response to environmental stress (boxed upward pointing arrow). Slp3 production is decreased in the yeast-to-hyphae transition (boxed downward pointing arrow). Figure adapted from references [5][6] and the ouresearchers' unpublished findings and created with Biorender.com.

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