Recombinant Ustilago maydis Presequence translocated-associated motor subunit PAM17, mitochondrial (PAM17)

What is PAM17 and what is its role in mitochondrial function?

PAM17 is a subunit of the presequence translocase-associated motor (PAM) of mitochondria. It functions as an integral component of the protein import machinery, specifically associated with the TIM23 complex. PAM17 is anchored in the mitochondrial inner membrane with its functional domain exposed to the matrix . The protein is essential for the proper functioning of the mitochondrial import motor that drives translocation of preproteins across the inner membrane. Studies in yeast have shown that PAM17 is specifically required for transport of proteins into the mitochondrial matrix but not for insertion of proteins into the inner membrane .

How is PAM17 synthesized and processed in cells?

PAM17 is synthesized as a precursor protein containing a cleavable presequence. Experimental evidence from yeast shows that the precursor migrates more slowly on SDS-PAGE than the mature form. When incubated with isolated mitochondria in the presence of a membrane potential (Δψ), the PAM17 precursor is processed to the mature-sized form. This processing is inhibited when the membrane potential is dissipated, confirming that PAM17 follows the classical import pathway for mitochondrial inner membrane proteins with matrix-exposed domains .

How does PAM17 interact with other components of the mitochondrial import machinery?

PAM17 plays a crucial role in organizing the architecture of the mitochondrial import motor. While PAM17 itself is not a direct component of the Pam16-Pam18 complex, it is required for the proper association of this complex with the TIM23 translocase. In pam17Δ mitochondria, the amount of Pam16 and Pam18 recovered with tagged Tim23 is significantly decreased compared to wild-type mitochondria, demonstrating that PAM17 facilitates or stabilizes the interaction between the Pam16-Pam18 module and the membrane-embedded TIM23 complex .

When analyzed by blue native polyacrylamide gel electrophoresis (BN-PAGE), PAM17 migrates in a band of approximately 50 kDa, separately from the Pam16-Pam18 complex. This indicates that while PAM17 influences the association of Pam16-Pam18 with the TIM23 complex, it does not form a stable subcomplex with these proteins .

What specific molecular mechanisms underlie PAM17's role in protein translocation?

How is PAM17 from Ustilago maydis structurally and functionally similar to or different from its homologs in other organisms?

While the search results don't provide specific information about PAM17 in Ustilago maydis, we can infer from fungal conservation patterns that as a basidiomycete, U. maydis likely possesses a PAM17 homolog functionally similar to that in ascomycetes like Saccharomyces cerevisiae. U. maydis has become an important model organism for studying biotrophic fungi, with extensive transcriptional profiling available for various developmental stages .

What are the most effective methods for isolating and characterizing recombinant PAM17?

Based on established protocols for mitochondrial proteins, effective isolation of recombinant PAM17 would involve:

• Expression system selection: Either heterologous expression in E. coli or homologous expression in U. maydis, with a purification tag (e.g., His-tag or FLAG-tag)

• Purification strategy:

  • Affinity chromatography using the added tag

  • For membrane proteins like PAM17, detergent solubilization is critical (typical detergents include n-dodecyl-β-D-maltoside or digitonin)

  • Size exclusion chromatography to ensure protein homogeneity

• Characterization techniques:

  • SDS-PAGE and Western blotting to confirm identity

  • Circular dichroism to assess secondary structure

  • BN-PAGE to analyze native complex formation (PAM17 migrates at approximately 50 kDa)

  • Protease protection assays to confirm topology (as demonstrated in the research where PAM17 was protected against protease treatment in mitoplasts but digested upon sonication)

What techniques are most appropriate for studying PAM17's interactions with other components of the import machinery?

Several complementary approaches can be used to study PAM17's protein-protein interactions:

• Co-immunoprecipitation: Using tagged versions of PAM17 or other TIM23-PAM components to pull down interacting partners. This approach has successfully demonstrated that PAM17 cofractionates with other PAM subunits and is present in the preprotein-TOM-TIM23-PAM supercomplex .

• Chemical crosslinking: To capture transient interactions within the dynamic import machinery.

• Yeast two-hybrid assays: For mapping specific interaction domains.

• In vitro binding assays: With purified components to determine direct interactions.

• BN-PAGE analysis: To study native complexes under various conditions, as demonstrated in studies where PAM17 was shown to migrate separately from the Pam16-Pam18 complex .

• Preprotein arrest technique: Where a preprotein containing a folded domain (DHFR stabilized by methotrexate) is accumulated across mitochondrial membranes, allowing isolation of import-active translocase complexes containing PAM17 .

How can one assess the functional impact of PAM17 mutations or deletions?

Based on established methodologies in the field, functional assessment of PAM17 variants would include:

• Growth phenotype analysis: Testing growth of cells expressing mutant PAM17 variants under different conditions, particularly on non-fermentable carbon sources where mitochondrial function is essential .

• In vitro import assays: Using isolated mitochondria to assess import efficiency of various substrates:

  • Matrix-targeted proteins (e.g., F1β-ATPase)

  • Inner membrane proteins with stop-transfer signals (e.g., cytochrome c1)

  • Model fusion proteins (e.g., b2(167)Δ-DHFR for matrix import)

• Membrane potential measurements: Using fluorescent dyes like DiSC3(5) to ensure that import defects are not secondary to membrane potential defects .

• Import motor activity assay: Using the two-step import protocol where a preprotein is first accumulated across both membranes, followed by Δψ dissipation and assessment of continued import driven solely by the import motor .

• Assembly analysis: Investigating how mutations affect the association of PAM17 with the TIM23 complex and the recruitment of other import motor components, particularly Pam16 and Pam18 .

What is known about the structure-function relationship of PAM17?

The current understanding of PAM17's structure-function relationship includes:

• Topology: PAM17 is anchored in the inner mitochondrial membrane with its functional domain exposed to the matrix. This has been experimentally verified through protease protection assays, where PAM17 remains protected against added protease in mitoplasts (like matrix-exposed Tim44) but becomes accessible to protease upon sonication to open the matrix .

• Domain organization:

  • N-terminal presequence (cleaved upon import)

  • Transmembrane domain(s) anchoring the protein in the inner membrane

  • Matrix-exposed functional domain that likely mediates interactions with other import motor components

• Functional elements:

  • Regions involved in facilitating the association of Pam16-Pam18 with the TIM23 complex

  • Potential sites for interaction with Tim17, as demonstrated by reduced copurification of PAM17 with tagged Tim23 in tim17-5 mutant mitochondria

How does the membrane potential (Δψ) influence PAM17 function in protein import?

PAM17 functions at the interface between the membrane potential (Δψ)-dependent and ATP-dependent phases of protein import:

• Processing of PAM17 itself: The import and processing of PAM17 precursor is Δψ-dependent, as demonstrated by inhibition of processing upon dissipation of the membrane potential .

• Role in motor activity: PAM17 is specifically required for the ATP-driven import motor activity that continues protein translocation after the initial Δψ-dependent step. In experimental settings where Δψ is dissipated after preprotein accumulation across both membranes, pam17Δ mitochondria show strongly reduced further translocation .

• Substrate specificity: The import of matrix proteins, which require both Δψ and import motor activity, is strongly impaired in pam17Δ mitochondria. In contrast, the import of inner membrane proteins with stop-transfer signals, which primarily depend on Δψ, remains efficient .

This suggests that PAM17 plays a critical role in coupling the Δψ-dependent initial translocation with the subsequent ATP-dependent motor-driven import phase.

How does PAM17 expression vary across different developmental stages in Ustilago maydis?

While the search results don't provide specific information about PAM17 expression patterns in U. maydis, we can note that comprehensive transcriptional profiling data exists for U. maydis throughout its developmental cycle. The GSE103876 dataset includes RNA-seq analysis of U. maydis during eight different stages of plant-associated development, including growth on the leaf surface, early colonization, tumor induction, and spore maturation .

This dataset could be analyzed to determine PAM17 expression patterns across these developmental stages, potentially revealing whether mitochondrial import processes are differentially regulated during the biotrophic lifecycle of U. maydis. Such analysis might indicate whether PAM17 expression is correlated with specific developmental transitions or metabolic adaptations during host colonization.

What evolutionary insights can be gained from comparing PAM17 across different fungal species?

Evolutionary analysis of PAM17 across fungal species could provide valuable insights into:

• Conservation patterns: Identifying highly conserved regions likely critical for function versus more variable regions that might reflect species-specific adaptations.

• Correlation with lifestyle: Comparing PAM17 from biotrophs like U. maydis with saprotrophs and other pathogens to identify potential adaptations related to different ecological niches.

• Co-evolution with interacting partners: Analyzing whether changes in PAM17 sequence correlate with changes in other components of the import machinery.

U. maydis, as a basidiomycete, represents a different fungal lineage from the well-studied ascomycete S. cerevisiae, potentially offering insights into the evolution of mitochondrial import mechanisms across major fungal groups .

What are the most promising approaches for elucidating PAM17's precise molecular function?

Based on current knowledge gaps, several approaches could advance our understanding of PAM17:

• High-resolution structural studies: Cryo-EM or X-ray crystallography of PAM17 alone and in complex with other import components would provide crucial insights into its molecular mechanism.

• Single-molecule techniques: To monitor the dynamics of protein translocation in real-time and determine how PAM17 influences this process.

• Systematic mutagenesis: To identify critical residues and domains required for PAM17's role in organizing the import motor.

• Interactome mapping: Using proximity labeling approaches like BioID to identify the full range of PAM17 interactions during different stages of protein import.

• In vitro reconstitution: Of minimal functional import systems to directly test PAM17's role in motor function.

How might understanding PAM17 function contribute to broader knowledge of mitochondrial disease mechanisms?

While not directly addressed in the search results, understanding PAM17 function has implications for mitochondrial diseases:

• Import deficiency diseases: Several human mitochondrial diseases involve defects in protein import machinery. Understanding the fundamental mechanisms of import motor function could provide insights into pathological processes.

• Therapeutic targeting: The protein import machinery represents a potential therapeutic target for mitochondrial disorders. Detailed knowledge of PAM17's role could inform the development of targeted interventions.

• Cellular stress responses: Mitochondrial protein import is regulated during various stress conditions. Understanding how PAM17 contributes to import motor function could illuminate how cells adapt mitochondrial function during stress.

• Aging mechanisms: Mitochondrial dysfunction is a hallmark of aging. The integrity of protein import pathways, including PAM17-dependent processes, may influence age-related decline in mitochondrial function.

The research on U. maydis PAM17 could provide a valuable comparative perspective on these processes, complementing studies in more traditional model systems.