Recombinant Transmembrane GTPase fzo-1 (fzo-1)

What is the primary function of transmembrane GTPase fzo-1?

Transmembrane GTPase fzo-1 (Fzo1 in yeast) belongs to the mitofusin family of dynamin-related GTPase proteins (DRPs) that mediate fusion of mitochondrial outer membranes. Unlike conventional GTPases that function as molecular switches cycling between inactive GDP-bound and active GTP-bound forms, fzo-1 activity occurs concomitantly with GTP hydrolysis. The protein assembles into homo-dimers, allowing membrane tethering, followed by GTP hydrolysis which enables subsequent steps in the fusion process .

Research has demonstrated that mitofusins are essential players in maintaining mitochondrial network morphology, which directly impacts cellular energetics and stress responses. The fusion cycle mediated by fzo-1 involves distinct steps including protein assembly, membrane tethering, GTP hydrolysis, and regulated protein degradation, making it a unique membrane fusion mechanism distinct from other cellular membrane fusion events .

How is the structure of fzo-1 organized to enable its function?

Fzo-1 has a complex multi-domain architecture that facilitates its membrane fusion activity. Full-atom homology modeling of Fzo1 (yeast homolog) has revealed several key structural features:

• A GTPase domain that binds GDP/GTP

• Multiple helical repeat domains (HRN, HR1, HR2) with complex organization

• Transmembrane (TM) domains that anchor the protein in the mitochondrial outer membrane

• Hinges that connect helical spans and likely have functional significance

The structural organization of fzo-1 is more complex than initially hypothesized. For example, the helical repeat (HR) domains are not merely continuous helices but are composed of several tandem helices separated by flexible hinges. This complex arrangement explains why mutations in certain regions dramatically affect protein function .

Recent structural modeling suggests that fzo-1 can adopt different conformations, including "stretched dimer" and "bent dimer" states, which likely represent different functional states during the fusion process . These conformational changes are thought to drive membrane remodeling during fusion.

What experimental systems are most suitable for studying fzo-1?

Several experimental systems have proven effective for investigating fzo-1 function:

For respiratory function assessment in yeast, researchers commonly use a comparative growth analysis on glucose versus glycerol-containing media. This approach has helped identify critical regions of fzo-1, such as the observation that deletion of residues 61-91 completely abolishes respiratory growth while stabilizing the protein .

Molecular dynamics simulations in a mixed lipid bilayer environment have been particularly useful for analyzing the stability of fzo-1 structural models, confirming the coherent and stable architecture of key domains including the coiled-coil domains HRN, HR1, HR2, and the TM dimer .

How does ubiquitylation regulate fzo-1 activity?

Ubiquitylation of fzo-1 represents a sophisticated regulatory mechanism that can either promote or inhibit mitochondrial fusion, depending on the specific modification pattern. Research has identified a complex regulatory system:

• Pro-fusion ubiquitylation: Attachment of ubiquitin chains to lysine 398 (requiring prior ubiquitylation of lysine 464) promotes fusion activity and increases protein stability .

• Anti-fusion ubiquitylation: Different ubiquitin modifications can inhibit fusion activity .

• Deubiquitylation control: A balanced system involving two deubiquitylating enzymes (DUBs):

  • Ubp12: Removes pro-fusion ubiquitin chains

  • Ubp2: Removes anti-fusion ubiquitin chains

• Degradation pathway: After completing its function in outer membrane fusion, fzo-1 undergoes Mdm30-dependent degradation .

This multilayered regulatory cascade is further controlled by Cdc48, which physically interacts with ubiquitylated fzo-1. Importantly, Cdc48 binding specifically recognizes the pro-fusion ubiquitylated forms of fzo-1, providing another layer of regulation . Mutations affecting this regulatory system significantly impact mitochondrial morphology and respiratory capacity.

What protein interactions are critical for fzo-1-mediated membrane fusion?

Fzo-1 operates through a network of protein interactions that coordinate mitochondrial outer membrane fusion. The key interactions include:

These interactions function in a sequential manner. First, Ugo1 facilitates GTP-dependent homo-dimerization of fzo-1. After membrane tethering and GTP hydrolysis, Mdm30-dependent ubiquitylation and subsequent degradation of fzo-1 complete the fusion process .

Importantly, Cdc48 interacts with both fzo-1 and Ubp2, orchestrating a multilayered cascade regulation culminating in fzo-1 ubiquitylation and mitochondrial fusion . Experimental evidence shows that overexpression of Ubp2 can rescue respiratory defects in cdc48-2 mutant cells, highlighting the physiological importance of this regulatory network .

How do conformational changes in fzo-1 drive membrane fusion?

Fzo-1 undergoes significant conformational changes that drive the membrane fusion process. Research using structural modeling and molecular dynamics simulations has revealed:

• Multiple conformational states: Fzo-1 can adopt at least two major conformations - a "stretched dimer" and a "bent dimer" configuration .

• GTP-dependent conformational switching: GTP binding and hydrolysis drive transitions between conformational states. The stretched dimer model is based on MFN1-MGD bound to GDP-BeF3− and BDLP bound to GMPPNP, while the bent dimer is modeled on GDP-AlF4− .

• Flexible hinges: The model identifies key hinges (notably hinges 1a and 1b) that connect helical spans and likely facilitate the large conformational rearrangements required during fusion .

The structural organization of fzo-1 includes complex helical repeat domains (HRN, HR1, HR2) that are not merely continuous helices but rather composed of several tandem helices. This arrangement contributes to the protein's ability to undergo significant conformational changes. In particular, helices α14 and α27, which are not defined by heptad periodicity, represent structural continuations of the HR1 and HR2 repeats separated by flexible hinges .

This configuration is comparable to the four-helix bundle observed in crystal structures of human Mfn1 fragments, where the C-terminal helix has been proposed to stabilize the bundle. Deletion experiments removing the last 24 residues of Fzo1 (Fzo1 Δ826–855) abolished mitochondrial fusion, confirming the functional importance of these structural elements .

What is the relationship between GTP binding/hydrolysis and fzo-1-mediated membrane tethering?

GTP binding and hydrolysis play distinct roles in fzo-1 function, orchestrating a sequence of events that enable mitochondrial membrane tethering and fusion:

• GTP binding: Essential for the initial assembly of fzo-1 into homo-dimers, which is a prerequisite for the subsequent steps in membrane fusion. This assembly process also depends on Ugo1 .

• Homo-dimer association: Following initial dimerization, fzo-1 homo-dimers further associate upon formation of mitochondrial contacts, enabling membrane tethering .

• GTP hydrolysis: After membrane tethering, GTP hydrolysis becomes necessary for fzo-1 ubiquitylation by the F-box protein Mdm30. This step differs from the classical GTPase cycle, as fzo-1 activity and downstream signaling occur concomitantly with GTP hydrolysis rather than through cycling between GDP/GTP-bound states .

• Structural basis: The GTPase domain of fzo-1 interacts with GDP through the G1 and G4 motifs, as revealed by structural modeling .

In experimental systems, mutations affecting GTP binding completely disrupt fzo-1 function. For instance, non-ubiquitylated Fzo1 variants like K464R show impaired interaction with regulatory proteins such as Cdc48, underscoring the connection between GTP-dependent activity and ubiquitylation pathways .

The complex relationship between GTP binding/hydrolysis and membrane fusion is further evidenced by structural models showing that nucleotide binding influences the conformation of fzo-1, with different conformational states observed when bound to different nucleotide analogs (e.g., GDP-BeF3− versus GDP-AlF4−) .

How does the Cdc48-deubiquitylase cascade regulate fzo-1 activity?

The Cdc48-deubiquitylase cascade represents a sophisticated regulatory mechanism for controlling fzo-1 function in mitochondrial fusion:

This cascade functions through several key mechanisms:

• Physical interaction: Cdc48 physically interacts with ubiquitylated fzo-1, showing preference for pro-fusion ubiquitylation forms. This interaction is impaired with non-ubiquitylated variants like Fzo1 K464R .

• Stabilization effect: Cdc48 maintains fzo-1 protein levels by protecting it from premature degradation. In cdc48-2 mutant cells, fzo-1 levels decrease due to increased proteasome-dependent turnover, which can be prevented by proteasome inhibition .

• Sequential regulation: Experimental evidence shows that deleting both UBP2 and UBP12 makes fzo-1 levels insensitive to CDC48 mutations, indicating that Cdc48 functions upstream of these deubiquitylases in the regulatory pathway .

• Rescue mechanisms: Overexpression of Ubp2 can rescue mitochondrial tubulation defects and improve respiratory growth in cdc48-2 mutant cells, demonstrating the physiological importance of this regulatory network .

The functional significance of this cascade extends beyond protein regulation to directly impact mitochondrial morphology. In cdc48-2 mutant cells, decreased fzo-1 levels correlate with fragmented mitochondria, while interventions that restore proper fzo-1 regulation (like UBP12 deletion or UBP2 overexpression) significantly improve mitochondrial tubulation and respiratory capacity .

What experimental approaches are most effective for site-directed mutagenesis studies of fzo-1?

Site-directed mutagenesis represents a powerful approach for investigating fzo-1 structure-function relationships. Based on structural models and functional studies, several effective experimental strategies have emerged:

• Targeting strategic regions:

  • GTPase domain: Mutations in GTP-binding motifs (G1 and G4) disrupt nucleotide binding and prevent initial dimerization .

  • Ubiquitylation sites: Mutations at lysine residues 398 and 464 prevent ubiquitylation and significantly alter protein function and stability .

  • Hinge regions: Mutations in flexible hinges that connect helical domains can disrupt conformational changes required for function .

  • Helical repeats: Targeted mutations in HRN, HR1 and HR2 domains can reveal their specific contributions to protein structure and function .

• Functional validation approaches:

  • Respiratory growth assays: Testing growth of yeast strains expressing fzo-1 mutants on glycerol-containing media to assess mitochondrial function .

  • Protein stability assessment: Analyzing steady-state levels of mutant proteins via Western blotting to determine effects on protein stability .

  • Protein interaction studies: Co-immunoprecipitation experiments to assess how mutations affect interactions with regulatory proteins like Cdc48, Ugo1, or Mdm30 .

  • Mitochondrial morphology analysis: Microscopy-based assessment of mitochondrial network structure in cells expressing mutant proteins .

• Deletion mutant analysis:
  Research has demonstrated the value of strategic deletion mutants in mapping functional domains. For example:

  • Deletion of the first 30 residues (1-30 fzo1Δ) showed normal growth on both glucose and glycerol media

  • Deletion of the first 60 residues (1-60 fzo1Δ) nearly completely abolished respiratory growth

  • Deletion of the first 91 residues (1-91 fzo1Δ) significantly increased protein stability

These findings help map the functional importance of specific regions and guide further targeted mutagenesis.

• Structure-guided mutagenesis:
  Modern approaches leverage structural models to design targeted mutations. For instance, the Fzo1 model simulated in membrane environments revealed long-distance contacts and residues participating in hinges that could be validated through site-directed mutagenesis . This approach has successfully identified structural determinants critical for protein function, including regions involved in GTPase domain-dependent rearrangements.

How can molecular dynamics simulations enhance our understanding of fzo-1 structure and function?

Molecular dynamics (MD) simulations provide valuable insights into fzo-1 structure and function that complement experimental approaches. Research applying MD techniques to fzo-1 has revealed:

• Conformational stability assessment:

  • Three extended 500-ns MD simulations in fully hydrated lipid membrane environments demonstrated the stability of the core Fzo1 model structure

  • Analysis of RMSD (Root Mean Square Deviation) values identified stable segments versus flexible regions

  • Secondary structure analysis confirmed retention of long α-helices, especially in coiled-coil domains HRN, HR1, HR2, and the TM dimer

• Identification of key structural elements:

  • MD simulations revealed that unstructured regions and portions with high flexibility (measured by RMSF - Root Mean Square Fluctuation) contribute to local structural deviations

  • Analysis of the transmembrane domain showed consistent stability across trajectories with an average RMSD value of 2.4 Å

• Validation through representative structures:

  • Cluster analysis of MD trajectories identified centroid structures representative of the protein's conformational ensemble

  • These representative structures can be used to guide experimental validation through targeted mutagenesis

• Insights into domain organization:
  MD simulations have revealed that the helical repeat (HR) domains have a more complex organization than initially hypothesized:

  • HRN, HR1 and HR2 are composed of several tandem helices rather than continuous helical structures

  • Previously uncharacterized regions, such as the α14 and α27 helices, contribute to protein function despite not being defined by heptad periodicity

  • These helices represent structural continuations of HR domains, separated by flexible hinges

• Membrane integration analysis:

  • MD simulations in lipid bilayers provide insights into how the transmembrane domains anchor and orient the protein within the mitochondrial outer membrane

  • These simulations help identify lipid-protein interactions that may influence function

This computational approach has been particularly valuable for integrating diverse experimental data and generating testable hypotheses about how structural features contribute to fzo-1's role in mitochondrial membrane fusion.

What experimental controls are essential when studying fzo-1 function in yeast systems?

When designing experiments to study fzo-1 function in yeast, several critical controls should be included to ensure reliable and interpretable results:

• Growth condition controls:

  • Compare growth on fermentable (glucose) versus non-fermentable (glycerol/lactate) carbon sources to assess respiratory capacity

  • Include temperature sensitivity tests (particularly at 37°C) as fzo-1 mutants often show temperature-dependent phenotypes

• Genetic complementation controls:

  • Use wild-type FZO1 expression as a positive control

  • Include fzo1Δ empty vector as a negative control

  • For specific mutations, include known functional/non-functional mutations as references

• Protein expression verification:

  • Monitor steady-state levels of mutant proteins compared to wild-type

  • Use stable, non-mitochondrial proteins as loading controls

  • For degradation studies, include proteasome inhibitor controls (e.g., MG132) to distinguish between synthesis and degradation effects

• Functional validation controls:
  When analyzing mitochondrial morphology:

  • Compare with wild-type tubular networks (positive control)

  • Compare with the fragmented phenotype of fzo1Δ cells (negative control)

  • Include other fusion/fission mutants to distinguish specific effects

For studies involving the Cdc48-deubiquitylase cascade, experimental evidence shows the importance of proper controls. For instance, when examining Cdc48's effect on Fzo1, researchers included analysis of Ubc6 (an ER membrane protein degraded via ERAD) as a control to demonstrate that Cdc48 regulates Fzo1 through a mechanism distinct from ERAD .

How can researchers effectively distinguish between different ubiquitylated forms of fzo-1?

Distinguishing between different ubiquitylated forms of fzo-1 is crucial for understanding its regulation, as these modifications can either promote or inhibit fusion. Several effective experimental approaches include:

• Mutational analysis of ubiquitylation sites:

  • K464R mutation prevents all forms of ubiquitylation

  • This can be used as a negative control to identify bands representing ubiquitylated species

• Manipulation of deubiquitylating enzymes (DUBs):

  • Expression of catalytically inactive Ubp2 C745S allows detection of anti-fusion ubiquitin forms

  • Deletion of UBP12 enhances detection of pro-fusion ubiquitin forms

  • Combined manipulation provides a comprehensive view of the ubiquitylation landscape

• Visualization techniques:

  • Western blotting with specific anti-ubiquitin antibodies

  • Pro-fusion ubiquitin forms appear as higher molecular weight bands (black arrows in published figures)

  • Anti-fusion ubiquitin forms (visible in the presence of Ubp2 C745S) appear as distinct bands (red arrows in published figures)

• Quantitative assessment:

  • Measure the relative abundance of ubiquitylated forms compared to unmodified protein

  • For example, expression of Ubp2 C745S increased Fzo1 ubiquitylation by 2.44 times in experimental systems

• Correlation with protein interactions:

  • Co-immunoprecipitation experiments can reveal how different ubiquitylation states affect interactions with regulatory proteins

  • For instance, despite increased ubiquitylation in the presence of Ubp2 C745S, Cdc48 binding to Fzo1 was not increased, indicating specificity for pro-fusion ubiquitin forms

These approaches have helped researchers establish that Cdc48 specifically recognizes pro-fusion ubiquitylated forms of Fzo1, similar to the specificity shown by Ubp12, highlighting the precision of this regulatory system .

What are the promising approaches for translating fzo-1 research to therapeutic applications?

While the provided search results focus primarily on basic research aspects of fzo-1/Fzo1, several promising research directions emerge that could lead to therapeutic applications:

• Targeting the ubiquitylation regulatory network:

  • The complex ubiquitylation/deubiquitylation system controlling fzo-1 offers multiple intervention points

  • Modulating specific DUBs (like Ubp2 or Ubp12 homologs in humans) might allow precise control of mitochondrial fusion rates

  • Research shows that overexpression of Ubp2 rescues respiratory defects in cdc48-2 mutants, suggesting a potential therapeutic approach

• Structure-based drug design:

  • The detailed structural models of fzo-1/Fzo1 provide templates for designing molecules that could modulate its activity

  • Small molecules targeting specific conformational states might allow controlled activation or inhibition of fusion

  • The identified hinge regions and GTP-binding pocket offer specific targets for drug development

• Exploiting the Cdc48-deubiquitylase cascade:

  • The multilayered regulatory system involving Cdc48 and deubiquitylating enzymes provides multiple intervention points

  • Research has shown that this cascade orchestrates fzo-1 ubiquitylation and mitochondrial fusion

  • Targeting specific components might allow fine-tuning of mitochondrial dynamics in disease states

• Leveraging conformational insights:

  • Understanding the relationship between different conformational states of fzo-1 and its function opens possibilities for designing modulators

  • The "stretched dimer" and "bent dimer" models provide templates for rational drug design approaches