Recombinant Saccharomyces cerevisiae Peroxisomal membrane protein PEX32 (PEX32)

What is PEX32 and what is its role in peroxisome biogenesis?

PEX32 belongs to the Pex23 family of peroxins, which are characterized by the presence of a DysF domain. These proteins play crucial roles in peroxisome biogenesis, including peroxisomal matrix protein import, membrane biogenesis, and organelle proliferation . In yeast species like Hansenula polymorpha, PEX32 is an ER protein required for associating peroxisomes to the ER . Deletion of PEX32 results in the loss of peroxisome-ER contacts, accompanied by defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning, and segregation .

What domains are present in PEX32 and what are their functions?

PEX32 contains two main functional domains:

• N-terminal transmembrane (TM) domain: Contains four predicted transmembrane helices that are responsible for sorting PEX32 to the ER. The second TM helix specifically harbors ER targeting information . This N-terminal domain is sufficient for the function of PEX32 in peroxisome biogenesis .

• C-terminal DysF domain: Required for concentrating PEX32 at ER-peroxisome contact sites and has the ability to bind to peroxisomes . The DysF domain was first identified in human dysferlin, which is important for fusion of vesicles with the sarcolemma at the site of muscle injury .

How does PEX32 deletion affect peroxisome biology?

Deletion of PEX32 has several significant impacts on peroxisome biology:

• Loss of peroxisome-ER contacts

• Strong reduction in peroxisome numbers

• Defects in peroxisomal matrix protein import

• Impaired membrane growth and organelle proliferation

• Abnormal positioning and segregation of peroxisomes

• Significantly reduced levels of peroxisomal protein Pex11

• Growth defects on glycerol/methanol media (in H. polymorpha)

How do the functions of PEX32 differ between yeast species?

Interesting species-specific differences have been observed in PEX32 function:

These differences highlight the importance of species-specific analysis when studying PEX32 function, as findings from one yeast species may not directly translate to others .

What is the relationship between PEX32 and Pex11?

A critical finding in PEX32 research is its relationship with Pex11:

• Pex11 levels are strongly reduced in pex32 cells .

• This reduction may explain the decreased peroxisome numbers in pex32 cells, which resembles the phenotype of cells lacking Pex11 .

• Pex11 contributes to Pex32-dependent peroxisome-ER contact formation. In cells lacking Pex11, accumulation of Pex32 at contact sites is lost, along with disruption of the contacts .

• Pex11 appears to be a general contact site resident protein, also being important for the formation of peroxisome-mitochondria contacts .

This relationship suggests a functional interplay between these two proteins in maintaining proper peroxisome-ER contacts and peroxisome abundance.

How do peroxisome-ER contact sites form and what is the role of PEX32?

Peroxisome-ER contact sites are critical membrane contact sites (MCS) where PEX32 plays a key role:

• PEX32 predominantly accumulates at peroxisome-ER contacts .

• The C-terminal DysF domain of PEX32 is required for concentrating it at these contact sites and has the ability to bind to peroxisomes .

• When expressed alone (without TM domains), the DysF domain partially localizes to peroxisomes, suggesting it can independently recognize peroxisomal binding partners .

• Pex11 contributes to PEX32-dependent peroxisome-ER contact formation .

• Defects caused by PEX32 deletion can be suppressed by introducing an artificial peroxisome-ER tether, confirming that PEX32 contributes to tethering peroxisomes to the ER .

How can researchers analyze the domain structure-function relationship of PEX32?

To analyze domain function in PEX32, researchers can employ these established methodologies:

• Truncation analysis: Create constructs containing different domains of PEX32 fused to a reporter (e.g., GFP) .

  • Full-length protein (control)

  • N-terminal domain with all four TMs

  • Individual TM domains

  • DysF domain alone

  • Various combinations of domains

• Complementation assays: Introduce truncated constructs into pex32 deletion strains and assess :

  • Restoration of peroxisome numbers

  • Growth on selective media (e.g., glycerol/methanol)

  • Peroxisome-ER contacts

• Protein stability analysis: Perform Western blot analysis to confirm expression levels and stability of truncated proteins .

• Subcellular localization: Use confocal microscopy with appropriate markers (e.g., Bip-mCherry-HDEL for ER, Pex14-mKate2 for peroxisomes) to determine localization of truncated proteins .

What microscopy techniques are most effective for studying PEX32 localization and peroxisome-ER contacts?

Several advanced microscopy techniques have proven valuable for PEX32 research:

• Confocal Laser Scanning Microscopy (CLSM) with Airyscan: Provides enhanced resolution for visualizing PEX32 at peroxisome-ER contact sites and distinguishing between different cellular compartments .

• Fluorescence co-localization analysis: Using differentially labeled markers (e.g., PEX32-GFP with Bip-mCherry-HDEL for ER or Pex14-mKate2 for peroxisomes) to quantify the degree of spatial overlap .

• Live-cell imaging: To monitor dynamic changes in PEX32 localization and peroxisome-ER contacts.

• Quantitative image analysis: For measuring:

  • Percentage of peroxisomes associated with the ER

  • Fluorescence intensity of PEX32 at contact sites

  • Number and size of peroxisomes in different genetic backgrounds

How can researchers quantitatively assess peroxisome function in PEX32 mutants?

To assess peroxisome function in PEX32 mutants, researchers can employ these quantitative approaches:

• Growth assays: Measuring growth rates on media requiring peroxisome function (e.g., glycerol/methanol media for methylotrophic yeasts) .

• Peroxisome quantification:

  • Count peroxisome numbers per cell using fluorescence microscopy

  • Measure peroxisome size distribution

  • Analyze peroxisome positioning and segregation during cell division

• Biochemical assays:

  • Measure activity of peroxisomal enzymes (e.g., alcohol oxidase in H. polymorpha)

  • Quantify protein levels of peroxisomal markers via Western blotting

  • Assess import efficiency of peroxisomal matrix proteins

How should researchers interpret changes in peroxisome numbers and morphology in PEX32 mutants?

Interpreting changes in peroxisome characteristics requires careful consideration:

• Reduced peroxisome numbers: May result from:

  • Direct effects of PEX32 on peroxisome biogenesis

  • Indirect effects via reduced Pex11 levels (which controls peroxisome proliferation)

  • Defects in peroxisome-ER contact sites

• Peroxisome clustering: Could indicate defects in:

  • Peroxisome positioning

  • Cytoskeletal interactions

  • Organelle inheritance during cell division

• Controls to include:

  • Wild-type strain

  • pex11 deletion strain (to compare phenotypes)

  • Complemented strain expressing full-length PEX32

• Quantification approach:

  • Count at least 100 cells per strain across 2-3 independent experiments

  • Use unbiased selection criteria

  • Apply appropriate statistical tests (ANOVA with post-hoc tests)

What methods can be used to analyze the interaction of PEX32 with other proteins at peroxisome-ER contact sites?

Several complementary approaches can be used to study PEX32 interactions:

• Proximity-based labeling:

  • BioID or TurboID fusion with PEX32 to identify nearby proteins

  • APEX2-based proximity labeling

• Co-immunoprecipitation:

  • Pull-down of tagged PEX32 followed by mass spectrometry

  • Western blot analysis for specific candidate interactors

• Yeast two-hybrid screening:

  • Using different domains of PEX32 as bait

  • Focused screens with peroxisomal or ER proteins

• Fluorescence-based interaction studies:

  • Fluorescence resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • Fluorescence correlation spectroscopy (FCS)

What are common challenges in expressing recombinant PEX32 and how can they be addressed?

Researchers often encounter these challenges when working with recombinant PEX32:

• Protein instability:

  • Problem: Truncated versions of PEX32 may show reduced stability, particularly when the first 31 N-terminal residues are deleted .

  • Solution: Monitor protein levels by Western blotting; consider using proteasome inhibitors; optimize expression conditions.

• Low expression levels:

  • Problem: Constructs like TM(III-IV) may show levels too low for reliable localization studies .

  • Solution: Use stronger promoters (e.g., PADH1 instead of the endogenous promoter); optimize codon usage; confirm transcript levels by RT-PCR.

• Mislocalization artifacts:

  • Problem: Overexpression or truncation may lead to artificial localization patterns.

  • Solution: Compare expression under endogenous and strong promoters; include appropriate controls; use multiple tagging strategies.

How can researchers distinguish between direct and indirect effects of PEX32 deletion?

Distinguishing direct and indirect effects requires careful experimental design:

• Use of artificial tethers:

  • Introducing artificial peroxisome-ER tethers in pex32 cells can help determine which phenotypes are directly related to loss of tethering versus other functions .

• Analysis of secondary effects:

  • The finding that Pex11 levels are reduced in pex32 cells suggests some phenotypes may be indirect consequences of reduced Pex11 .

  • Test whether restoring Pex11 levels (e.g., by overexpression) rescues specific phenotypes.

• Acute inactivation approaches:

  • Use conditional alleles or rapid protein degradation systems instead of gene deletions.

  • This can help distinguish primary effects from adaptive responses.

What are promising areas for future research on PEX32?

Several exciting research directions emerge from current PEX32 findings:

• Structural biology approaches:

  • Determine the 3D structure of the DysF domain and its interaction with binding partners

  • Investigate the membrane topology of the TM domains

• Species-specific differences:

  • Further explore why the DysF domain is essential in some yeast species but not in H. polymorpha

  • Identify compensatory mechanisms that might exist in different species

• Regulation of peroxisome-ER contacts:

  • Investigate how contacts are regulated in response to metabolic conditions

  • Determine if post-translational modifications of PEX32 control its activity

• PEX32-Pex11 relationship:

  • Elucidate the molecular mechanism by which PEX32 influences Pex11 levels

  • Investigate whether this relationship is conserved across species

These future directions will help unravel the complex roles of PEX32 in peroxisome biogenesis and organelle contact site formation, contributing to our fundamental understanding of subcellular compartmentalization.