Recombinant Saccharomyces cerevisiae Presequence translocated-associated motor subunit PAM17, mitochondrial (PAM17)

What is the basic structure and localization of PAM17 in Saccharomyces cerevisiae?

PAM17, encoded by the open reading frame YKR065c in S. cerevisiae, consists of 197 amino acid residues including a mitochondrial presequence and two predicted hydrophobic segments of sufficient length to function as transmembrane segments. The protein is synthesized with a cleavable N-terminal presequence that directs its import into mitochondria. Biochemical analyses demonstrate that PAM17 is anchored in the mitochondrial inner membrane and is exposed to the matrix side. This topology was confirmed through protease protection assays, where PAM17 remained protected against added protease in mitoplasts (mitochondria with opened intermembrane space) similar to the matrix-exposed Tim44, but was digested upon sonication that disrupts the matrix compartment .

When subjected to alkaline extraction at pH 11.5, PAM17 fractionates with the membrane pellet like integral membrane proteins Tim23 and Tim50, while peripheral membrane proteins like Tim44 are extracted into the supernatant, confirming its identity as an integral membrane protein .

What is the primary function of PAM17 in mitochondrial protein import?

PAM17 functions as a component of the presequence translocase-associated motor (PAM) that drives the completion of preprotein translocation into the mitochondrial matrix. While not directly participating in preprotein binding, PAM17 plays a crucial architectural role in organizing the import motor. Specifically, PAM17 is required for the formation of a stable complex between the cochaperones Pam16 and Pam18, and promotes the association of this Pam16-Pam18 complex with the presequence translocase (TIM23 complex) .

Functional studies with mitochondria lacking PAM17 (pam17Δ) show that these organelles are selectively impaired in the import of matrix proteins and in generating the import-driving activity of PAM. This is evidenced by experiments measuring the Δψ-independent motor activity with a two-membrane-spanning preprotein, where pam17Δ mitochondria showed significantly reduced protease resistance of intermediate-sized b2(220)-DHFR compared to wild-type mitochondria, indicating impaired import-driving activity of PAM .

How can recombinant PAM17 be isolated and purified for biochemical studies?

For isolation and purification of PAM17, researchers typically employ affinity chromatography approaches utilizing tagged versions of TIM23 complex components. In published studies, the TIM23 complex and associated PAM were purified from S. cerevisiae mitochondria carrying a protein A tag at Tim23. This approach allowed identification of PAM17, which migrates similarly to Tim17 on most gel systems but can be resolved using high-resolution gel electrophoresis .

For characterizing recombinant PAM17:

• Clone the YKR065c gene into an appropriate expression vector

• Express the protein in a suitable host system (E. coli or yeast expression systems)

• Include affinity tags (His, GST, or Protein A) to facilitate purification

• Perform affinity chromatography under conditions that maintain protein structure

• Verify purity by SDS-PAGE and identity by mass spectrometry

• Confirm proper folding through functional assays

When studying PAM17 in its native context, researchers should consider that it comigrates with Tim17 on standard gel systems, necessitating high-resolution gel electrophoresis for proper separation and identification .

What experimental approaches are used to study PAM17's role in mitochondrial protein import?

Several complementary experimental approaches are employed to study PAM17's role in mitochondrial protein import:

• In vitro import assays: Using isolated mitochondria and radiolabeled preproteins. This approach can demonstrate the selective impairment of matrix protein import in pam17Δ mitochondria.

• Two-step import assays: To specifically measure PAM-dependent import activity, researchers:

  • First accumulate preproteins across both membranes

  • Then dissipate the membrane potential (Δψ) with valinomycin

  • Measure protease resistance of the accumulated intermediates

  This method revealed that in wild-type mitochondria, a significant fraction of b2(220)-DHFR was protected from protease, while in pam17Δ mitochondria most of the intermediate-sized b2(220)-DHFR was degraded, demonstrating PAM17's requirement for import-driving activity .

• Crosslinking studies: Using chemical crosslinkers to identify interaction partners. This approach showed altered crosslinking patterns of Tim44, Tim16, and Tim23 in the absence of Pam17, indicating conformational changes in the TIM23 complex .

• Blue native PAGE (BN-PAGE): To analyze intact protein complexes. This revealed that PAM17 migrates in a BN-PAGE band of about 50 kDa, separately from the Pam16-Pam18 complex .

• Immunoprecipitation: Using antibodies against TIM23 complex components to precipitate associated proteins and demonstrate interactions within the complex .

How does PAM17 regulate the Pam16-Pam18 complex assembly and function?

PAM17 plays a critical role in regulating the Pam16-Pam18 complex, though it is not a constituent subunit of this complex. Research indicates that PAM17 is required for the formation of a stable complex between the cochaperones Pam16 and Pam18, which together regulate the ATPase activity of mtHsp70 at the inner membrane translocation site.

In mitochondria lacking PAM17 (pam17Δ), blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis shows a substantial reduction in the amount of Pam16-Pam18 complex. Importantly, the residual Pam16-Pam18 complex in pam17Δ mitochondria showed the same mobility on BN-PAGE as the wild-type complex, indicating that PAM17 itself is not a genuine subunit of this complex but rather facilitates its assembly or stability. This is supported by the observation that PAM17 migrates in a BN-PAGE band of about 50 kDa and thus separately from the Pam16-Pam18 complex .

The exact molecular mechanism by which PAM17 promotes the formation of the Pam16-Pam18 complex remains to be fully elucidated, but evidence suggests it may involve architectural changes in the TIM23 complex that create a favorable environment for Pam16-Pam18 association.

What is the functional relationship between PAM17 and mtHsp70 in protein translocation?

While PAM17 does not directly interact with mtHsp70, it indirectly influences mtHsp70 function through its effects on the Pam16-Pam18 complex. The preprotein-binding matrix heat shock protein 70 (mtHsp70) is a central component of the import motor, providing the ATP-dependent pulling force for preprotein translocation. Its activity at the inner membrane is regulated by the J-complex consisting of Pam16-Pam18.

PAM17 contributes to mtHsp70 function by ensuring proper organization of the Pam16-Pam18 complex, which in turn regulates the ATPase activity of mtHsp70. In pam17Δ mitochondria, the import-driving activity of PAM is significantly reduced, as demonstrated by experiments measuring the Δψ-independent motor activity with a two-membrane-spanning preprotein .

The current model suggests a cascade of regulation:

• PAM17 promotes the formation of a stable Pam16-Pam18 complex

• Pam16-Pam18 complex properly associates with the presequence translocase

• This association enables precise regulation of mtHsp70 ATPase activity

• Regulated mtHsp70 activity drives preprotein translocation into the matrix

This regulatory cascade explains why pam17Δ mitochondria are selectively impaired in the import of matrix proteins while insertion of inner membrane proteins remains largely unaffected .

How does PAM17 interact with the TIM23 complex and what is its relationship with Tim21?

PAM17 interacts with the Tim17-Tim23 core of the TIM23 complex, playing a role in its organization and function. Intriguingly, PAM17 and Tim21 have been found to have antagonistic effects on the TIM23 complex. Experimental evidence from overexpression studies demonstrates this functional connection:

• Overexpression of Tim21 leads to decreased efficiency of Tim23 crosslinking to Pam17 and increased efficiency of crosslinking between Tim23 molecules.

• Overexpression of Pam17 counteracts this effect. In mitochondria where both proteins are overexpressed, adducts of two Tim23 molecules are virtually absent, and the intensity of the Tim23-Pam17 adduct is restored to almost wild-type levels.

• Immunoprecipitation experiments with digitonin-solubilized mitochondria containing overexpressed Pam17, Tim21, or both showed that:

  • Overexpression of Pam17 reduces the amounts of Tim21 precipitated with the TIM23 complex

  • Overexpression of Tim21 leads to virtually complete removal of Pam17 from the TIM23 complex

  • When both are overexpressed, increased levels of Pam17 lead to partial removal of overexpressed Tim21 from the complex

These findings suggest that Tim21 and Pam17 do not bind to the TIM23 complex simultaneously and have opposing functions in regulating the conformation and activity of the translocase .

What other protein interactions are critical for PAM17 function?

Beyond its interactions with the Tim17-Tim23 core of the TIM23 complex and its functional antagonism with Tim21, PAM17 participates in a network of interactions that facilitate mitochondrial protein import. Key interactions include:

The evidence suggests that PAM17 functions primarily as an architectural organizer within the import machinery rather than directly participating in preprotein binding or translocation. This organizing role makes PAM17 essential for the proper assembly and function of the protein import machinery, particularly for the import of matrix-targeted proteins .

What phenotypes are observed in PAM17 deletion mutants and how can they be used in research?

PAM17 deletion mutants (pam17Δ) display several characteristic phenotypes that make them valuable tools for studying mitochondrial protein import:

These phenotypes make pam17Δ mutants valuable experimental tools for:

• Distinguishing between matrix protein import and inner membrane protein insertion pathways

• Analyzing the specific contribution of the import motor to protein translocation

• Studying the architectural organization of the TIM23 complex

• Investigating genetic interactions with other components of the mitochondrial protein import machinery

How can researchers effectively perform comparative studies between wild-type and mutant PAM17?

To effectively compare wild-type and mutant PAM17 variants, researchers should implement a systematic approach combining multiple methods:

• Creation of expression constructs:

  • Develop a series of PAM17 variants including wild-type, point mutations, truncations, and domain swaps

  • Use consistent expression vectors with identical promoters and tags

  • Generate corresponding plasmids for expression in both E. coli (for in vitro studies) and yeast (for in vivo complementation)

• Complementation studies in pam17Δ strains:

  • Transform pam17Δ yeast with plasmids expressing different PAM17 variants

  • Assess growth rates under various conditions (temperature, carbon source)

  • Quantify respiratory competence through oxygen consumption measurements

  • Evaluate mitochondrial morphology and distribution

• In vitro import assays using isolated mitochondria:

  • Prepare mitochondria from wild-type, pam17Δ, and complemented strains

  • Use radiolabeled matrix-targeted preproteins as substrates

  • Quantify import rates and efficiencies under standard conditions

  • Perform two-step import assays to specifically measure PAM activity

  • Compare results using various preproteins with different targeting signals

• Structural analysis of TIM23-PAM complex:

  • Use BN-PAGE to analyze complex assembly

  • Perform crosslinking studies to detect conformational changes

  • Conduct co-immunoprecipitation experiments to assess protein interactions

  • Compare Pam16-Pam18 complex formation efficiency

• Data analysis and interpretation:

  • Quantify results from multiple independent experiments

  • Perform statistical analysis to determine significance

  • Create structure-function relationships based on mutation analyses

  • Consider developing mathematical models of protein import kinetics

How does PAM17 participate in remodeling of the TIM23 complex during different translocation modes?

PAM17 appears to play a significant role in the dynamic remodeling of the TIM23 complex during different translocation modes. Research suggests that the TIM23 complex can adopt different conformations depending on whether it's engaged in translocating proteins across the inner membrane into the matrix or inserting proteins into the inner membrane.

Evidence for PAM17's role in this remodeling includes:

• Altered crosslinking patterns of Tim23, Tim44, and Tim16 in the absence of PAM17, suggesting changes in the conformation of both the membrane-integrated part of the complex and the import motor .

• The antagonistic relationship between PAM17 and Tim21, where:

  • Tim21 is associated with the sorting mode of the TIM23 complex

  • PAM17 is associated with the motor-active mode

  • Overexpression experiments show they have opposing effects on Tim23 interactions

• Observations that PAM17 and Tim21 appear not to bind simultaneously to the TIM23 complex, suggesting they may represent or promote different functional states of the translocase .

Current models propose that PAM17 helps stabilize a conformation of the TIM23 complex that facilitates complete translocation into the matrix, while Tim21 promotes a conformation favoring lateral release into the inner membrane. This dynamic remodeling ensures proper sorting of preproteins to their correct mitochondrial subcompartments, with PAM17 playing a key role in the architecture that supports matrix-targeted import .

What experimental approaches can be used to investigate the structural dynamics of PAM17 during protein translocation?

Investigating the structural dynamics of PAM17 during protein translocation requires sophisticated biophysical and biochemical approaches:

• Site-specific crosslinking with photo-activatable or chemical crosslinkers:

  • Incorporate crosslinking agents at specific positions in PAM17

  • Trigger crosslinking at different stages of preprotein translocation

  • Identify crosslinked partners using mass spectrometry

  • Map interaction surfaces that change during the translocation process

• FRET (Förster Resonance Energy Transfer) analysis:

  • Create PAM17 variants labeled with fluorescent donor probes

  • Label potential interaction partners with acceptor probes

  • Measure changes in FRET efficiency during translocation

  • Calculate distance changes between proteins during the import process

• Single-particle cryo-electron microscopy:

  • Isolate intact TIM23-PAM complexes at different stages of translocation

  • Perform structural analysis to determine conformational changes

  • Create 3D reconstructions of the complex with and without engaged preproteins

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Expose the complex to deuterium under various conditions

  • Analyze the rate of hydrogen-deuterium exchange

  • Identify regions of PAM17 that undergo conformational changes during activity

• Real-time kinetic studies:

  • Develop assays that can monitor conformational changes in millisecond timeframes

  • Correlate these changes with stages of preprotein translocation

  • Determine the sequence and timing of events during protein import

• Molecular dynamics simulations:

  • Create computational models of PAM17 within the TIM23 complex

  • Simulate conformational changes during preprotein engagement

  • Generate testable hypotheses about dynamic structural rearrangements

These approaches, particularly when used in combination, would provide unprecedented insights into how PAM17 functions dynamically during the protein translocation process and how it contributes to the architectural remodeling of the TIM23 complex .