Recombinant Human ATP-dependent zinc metalloprotease YME1L1 (YME1L1)

What is YME1L1 and what are its primary functions in mitochondria?

YME1L1 (Yeast Mitochondrial Escape 1-Like 1) is a nuclear genome-encoded ATP-dependent zinc metalloprotease embedded in the inner mitochondrial membrane (IM) with its protease domain facing the intermembrane space (IMS), classifying it as an i-AAA protease . Initially identified in yeast as a gene product that elevates mitochondrial DNA migration to the nucleus, YME1L1 plays critical roles in:

• Degradation of damaged or misfolded proteins in the IM and IMS

• Processing of regulatory proteins like OPA1 that control mitochondrial dynamics

• Maintenance of mitochondrial proteostasis under stress conditions

• Regulation of mitochondrial protein import machinery

YME1L1's proteolytic activity is essential for maintaining proper mitochondrial function, as evidenced by the respiratory growth impairment in yeast lacking the yme1 gene .

How is YME1L1 imported into mitochondria and processed?

YME1L1 contains an N-terminal mitochondrial targeting sequence (MTS) that directs the protein to mitochondria. The import process follows these key steps:

• The precursor protein is synthesized in the cytosol with its MTS

• YME1L1 is imported into mitochondria using the TIM23 translocase that contains the ROMO1 subunit

• The MTS is cleaved by mitochondrial processing peptidase (MPP)

• The mature protein then assembles into its functional homo-oligomeric complex in the inner membrane

Interestingly, ROMO1 is specifically required for YME1L1 import, and YME1L1 itself regulates ROMO1 levels through degradation, creating a self-regulatory mechanism . This relationship creates a feedback loop where YME1L1 can influence its own import process.

What is the structure of YME1L1 and how does it function mechanistically?

YME1L1 forms a homo-oligomeric complex (~63 kDa per monomer) in the inner mitochondrial membrane . The protein contains:

• An N-terminal mitochondrial targeting sequence (cleaved during import)

• A transmembrane domain anchoring it in the inner membrane

• An AAA+ ATPase domain that provides energy for substrate unfolding

• A metalloprotease domain containing the zinc-binding motif for proteolysis

The functional YME1L1 assembles into a hexameric ring structure, with the proteolytic active sites facing the intermembrane space. Substrate degradation by YME1L1 follows these mechanistic steps:

• Recognition of accessible signal sequences on substrate proteins

• ATP-dependent unfolding of substrate proteins

• Translocation of unfolded proteins through the central pore

• Proteolytic degradation of substrates

Importantly, YME1L1 can processively unfold substrate proteins with substantial thermodynamic stabilities and discriminates between degradation signals based on amino acid composition .

What are the validated substrates of YME1L1?

YME1L1 has multiple confirmed substrates in the mitochondrial inner membrane and intermembrane space:

Studies of YME1L1-depleted cells reveal that this protease also influences levels of multiple components of the electron transport chain, particularly Complex IV subunits and assembly factors .

How does YME1L1 recognize its substrates?

YME1L1 substrate recognition follows specific principles identified through both in vivo and in vitro studies:

• Substrates must present an accessible signal sequence; degradation is not initiated simply by substrate unfolding

• YME1L1 discriminates between degradation signals based on amino acid composition, suggesting sequence-specific recognition in mitochondrial proteostasis

• The protease typically recognizes exposed domains or loops in the intermembrane space

• Protein damage signals, such as oxidative modification or misfolding, may enhance recognition

Experimental approaches using engineered proteases with soluble hexameric coiled coils replacing the transmembrane domain have enabled detailed in vitro studies of substrate recognition mechanisms . These studies have shown that specific amino acid sequences, rather than just structural instability, are critical for YME1L1 substrate selection.

How is YME1L1 activity regulated in response to cellular stress?

YME1L1 activity is dynamically regulated in response to various cellular stressors:

• Metabolic stress regulation:

  • Upon inhibition of mTORC1, a lipid signaling cascade is initiated via LIPIN1 (a PA phosphatase)

  • This leads to decreased phosphatidylethanolamine (PE) levels in the mitochondrial inner membrane

  • Reduced PE promotes proteolysis by YME1L1, enabling rewiring of the mitochondrial proteome

• Hypoxia and nutrient starvation:

  • YME1L1 participates in adapting the mitochondrial proteome to oxygen and nutrient availability

  • This adaptation involves selective degradation of specific substrates to optimize mitochondrial function under stress

• Self-regulation:

  • YME1L1 regulates its own import through the degradation of ROMO1, a component of the TIM23 complex specifically required for YME1L1 import

  • This creates a feedback mechanism balancing YME1L1 levels in mitochondria

This dynamic regulation allows YME1L1 to serve as a key mediator of mitochondrial adaptation to changing cellular conditions.

How does YME1L1 regulate mitochondrial fusion and fission?

YME1L1 plays a central role in regulating mitochondrial dynamics through its proteolytic processing of OPA1 (Optic Atrophy 1), a dynamin-like GTPase that controls mitochondrial fusion:

• OPA1 exists in long (L-OPA1) and short (S-OPA1) forms

• YME1L1 mediates the proteolytic processing of L-OPA1 to generate S-OPA1 under normal conditions

• Loss of YME1L1 accelerates OMA1-dependent L-OPA1 cleavage, resulting in:

  • Accumulation of S-OPA1

  • Increased mitochondrial fission

  • Mitochondrial network fragmentation

Studies in patient fibroblasts carrying YME1L1 mutations and YME1L1-deficient cellular models consistently show altered mitochondrial morphology with excessive fragmentation, demonstrating the critical importance of this protease in maintaining proper mitochondrial network structure .

What is the relationship between YME1L1, OPA1, and mitochondrial disease?

The functional interaction between YME1L1 and OPA1 has significant implications for mitochondrial diseases:

• OPA1 mutations cause dominant optic atrophy, the most common form of hereditary optic neuropathy

• YME1L1 mutations cause a mitochondriopathy with infantile-onset developmental delay, muscle weakness, ataxia, and optic nerve atrophy

• Both conditions feature optic nerve involvement, highlighting the importance of proper OPA1 processing in maintaining optic nerve health

In patients with YME1L1 mutations, the dysregulation of OPA1 processing contributes to the disease pathology through:

• Disrupted mitochondrial fusion and fission balance

• Compromised mitochondrial function in high-energy demanding tissues

• Impaired mitochondrial quality control

This relationship underscores the importance of precise proteolytic processing in maintaining mitochondrial homeostasis and preventing disease .

What experimental approaches can be used to study YME1L1's role in mitochondrial dynamics?

Several methodological approaches have proven effective for studying YME1L1's impact on mitochondrial dynamics:

• Live-cell imaging techniques:

  • Fluorescent labeling of mitochondria using MitoTracker dyes or mitochondria-targeted fluorescent proteins

  • Time-lapse microscopy to track mitochondrial fusion and fission events

  • Quantification of mitochondrial morphology (length, number, interconnectivity)

• Biochemical analysis of OPA1 processing:

  • Western blotting to monitor L-OPA1 and S-OPA1 ratios

  • In vitro proteolytic assays using recombinant YME1L1 and OPA1 substrates

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated genome editing to create YME1L1 knockouts

  • shRNA or siRNA-mediated silencing for transient depletion

  • Expression of mutant YME1L1 variants to study specific functional domains

• Mitochondrial functional analysis:

  • Measurement of mitochondrial membrane potential

  • Assessment of reactive oxygen species production

  • Quantification of ATP synthesis

  • Analysis of respiratory chain complex activities

These methods have been successfully employed to demonstrate that YME1L1 depletion disrupts mitochondrial function, causing mitochondrial depolarization, ROS generation, lipid peroxidation, and reduced ATP production .

What mutations in YME1L1 are associated with human pathologies?

Several pathogenic mutations in YME1L1 have been identified in human disease:

• The homozygous missense mutation R149W in the mitochondrial pre-sequence has been found in a consanguineous pedigree of Saudi Arabian descent . This mutation:

  • Is located in a highly conserved region in the mitochondrial pre-sequence

  • Inhibits cleavage of YME1L1 by the mitochondrial processing peptidase

  • Results in rapid degradation of YME1L1 precursor protein

  • Causes a novel mitochondriopathy with optic nerve atrophy

• Additional mutations may exist but remain to be fully characterized in the literature

The R149W mutation specifically affects a critical arginine residue that appears to function as part of the MPP recognition and cleavage site in YME1L1 . Studies involving mutagenesis of various arginine residues in the N-terminal region of YME1L1 have confirmed the importance of this site for proper processing of the protein.

How does YME1L1 dysfunction contribute to mitochondrial diseases?

YME1L1 dysfunction leads to mitochondrial disease through several mechanisms:

• Impaired proteostasis:

  • Accumulation of damaged or misfolded proteins in the inner membrane and intermembrane space

  • Disruption of protein quality control systems

• Altered mitochondrial dynamics:

  • Abnormal processing of OPA1 leading to mitochondrial fragmentation

  • Compromised mitochondrial fusion capability

• Dysregulated protein import:

  • Altered levels of TIM23 complex components

  • Inefficient import of nuclear-encoded mitochondrial proteins

• Metabolic consequences:

  • Impaired respiratory chain function

  • Reduced ATP production

  • Increased reactive oxygen species generation

These mechanisms collectively manifest as a mitochondriopathy with infantile-onset developmental delay, muscle weakness, ataxia, and optic nerve atrophy in patients with YME1L1 mutations .

What is the role of YME1L1 in cancer progression?

Recent research has uncovered significant connections between YME1L1 and cancer biology:

• YME1L1 expression in nasopharyngeal carcinoma (NPC):

  • YME1L1 is significantly upregulated in NPC tissues from patients

  • Elevated expression is observed across various primary human NPC cells

  • Low expression is maintained in adjacent normal tissues and primary nasal epithelial cells

• Functional impact of YME1L1 in cancer cells:

  • Genetic silencing or knockout of YME1L1 in NPC cells leads to:

    • Disrupted mitochondrial function

    • Mitochondrial depolarization

    • Increased ROS generation and lipid peroxidation

    • Reduced ATP production

    • Impaired cell viability, proliferation, and migration

    • Enhanced apoptosis activation

• Signaling pathway involvement:

  • YME1L1 plays a pivotal role in Akt-mTOR activation in NPC cells

  • Akt-S6K phosphorylation decreases upon YME1L1 depletion

  • Constitutively-active Akt1 mutant can rescue the inhibitory effects of YME1L1 silencing

• In vivo evidence:

  • Intratumoral administration of YME1L1-shRNA-expressing AAV curtails NPC xenograft growth in nude mice

These findings suggest YME1L1 as a potential therapeutic target in certain cancers, where its inhibition could disrupt mitochondrial function and suppress tumor growth.

What are effective methods for producing and purifying recombinant YME1L1?

Producing functional recombinant YME1L1 has been challenging due to its transmembrane domain. Recent advances have enabled successful approaches:

• Engineered soluble constructs:

  • Replacement of the transmembrane domain with a soluble hexameric coiled coil

  • This approach produces active YME1L1 hexamers that can be studied in vitro

  • Enables detailed biochemical and structural investigations

• Expression systems:

  • Bacterial expression systems (E. coli) for the soluble domains

  • Insect cell systems for full-length or modified constructs

  • Cell-free expression systems combined with recombinant MPP for processing studies

• Purification strategies:

  • Affinity chromatography using His-tags or other affinity tags

  • Ion exchange chromatography

  • Size exclusion chromatography to isolate properly assembled hexameric complexes

• Functional validation:

  • ATP hydrolysis assays to confirm ATPase activity

  • Proteolytic assays using known substrate peptides

  • Structural characterization to confirm proper oligomerization

These approaches have enabled researchers to overcome the inherent difficulties in working with membrane-anchored proteases and have facilitated detailed mechanistic studies of YME1L1 function .

What methodologies are recommended for studying YME1L1 substrate specificity?

Several complementary approaches can be employed to study YME1L1 substrate specificity:

• In vitro proteolytic assays:

  • Using purified recombinant YME1L1 (engineered soluble variants)

  • Testing candidate substrate proteins or peptides

  • Analyzing degradation products by SDS-PAGE, western blotting, or mass spectrometry

  • Determining kinetic parameters of proteolysis

• Substrate trap approaches:

  • Using catalytically inactive YME1L1 mutants to trap substrates

  • Co-immunoprecipitation followed by mass spectrometry identification

• Comparative proteomics:

  • Quantitative comparison of protein abundance in YME1L1-deficient versus control cells

  • Analysis of mitochondrial protein aggregates in YME1L1-deficient models

  • Stable isotope labeling approaches to track protein turnover rates

• Substrate signal sequence analysis:

  • Systematic mutagenesis of potential degradation signals in known substrates

  • Creation of fusion proteins with candidate degradation signals

  • Bioinformatic prediction of potential recognition motifs

Through these methods, researchers have determined that YME1L1 discriminates between degradation signals based on amino acid composition and requires substrates to present accessible signal sequences rather than simply relying on substrate unfolding .

How can YME1L1 function be assessed in cellular and animal models?

Several approaches have proven effective for evaluating YME1L1 function in biological systems:

• Cellular models:

  • CRISPR/Cas9-mediated genome editing to create YME1L1 knockouts

  • shRNA or siRNA-mediated silencing for transient depletion

  • Patient-derived fibroblasts harboring YME1L1 mutations

  • Rescue experiments with wild-type or mutant YME1L1 constructs

• Functional assays:

  • Mitochondrial morphology assessment using fluorescence microscopy

  • Western blot analysis of YME1L1 substrates (especially OPA1 processing)

  • Mitochondrial membrane potential measurements

  • ROS production quantification

  • ATP synthesis assays

  • Cell proliferation and viability assessments

  • Apoptosis detection methods

• Animal models:

  • YME1L1 knockout or knockdown models

  • Introduction of human disease-associated mutations

  • Tissue-specific deletion using conditional knockout strategies

  • AAV-mediated gene delivery for in vivo manipulation

• Phenotypic analyses:

  • Histological examination of tissues (particularly optic nerve)

  • Electron microscopy of mitochondrial ultrastructure

  • Behavioral assessments for neurological phenotypes

  • Metabolic characterization

These approaches have been successfully used to demonstrate that YME1L1 depletion causes mitochondrial dysfunction, affects cellular viability and proliferation, and can suppress tumor growth in xenograft models .

How is YME1L1 activity regulated by lipid environments and metabolic states?

Recent research has uncovered sophisticated regulatory mechanisms linking YME1L1 activity to cellular lipid metabolism and metabolic signaling:

• mTORC1-LIPIN1-YME1L1 axis:

  • Inhibition of mTORC1 activates the PA phosphatase LIPIN1

  • LIPIN1 activation depletes phosphatidic acid (PA) levels

  • This reduces synthesis of phosphatidylcholine (PC), phosphatidylserine (PS), and eventually phosphatidylethanolamine (PE)

  • Decreased PE levels in the mitochondrial inner membrane enhance YME1L1 proteolytic activity

• Positive feedback mechanism:

  • Activated YME1L1 degrades PRELI-like proteins involved in PS import for PE synthesis

  • This further reduces PE levels, creating a positive feedback loop

  • The outcome is continued upregulation of YME1L1 proteolysis and remodeling of the mitochondrial proteome

• Functional consequences:

  • This mechanism allows YME1L1 to participate in adapting mitochondrial function to nutrient availability

  • Under starvation conditions, YME1L1 helps rewire the mitochondrial proteome

  • YME1L1 may inhibit mitochondrial protein import during metabolic stress

This regulatory system demonstrates how YME1L1 functions as a sensor of cellular metabolic state, adjusting mitochondrial proteostasis to match nutrient availability and energy demands.

What is the interplay between YME1L1 and other mitochondrial proteases?

YME1L1 functions within a complex network of mitochondrial proteases that cooperatively maintain mitochondrial proteostasis:

• YME1L1 and OMA1 in OPA1 processing:

  • YME1L1 processes long OPA1 (L-OPA1) to generate a subset of short OPA1 (S-OPA1) forms

  • OMA1, another mitochondrial protease, can also cleave L-OPA1 at different sites

  • Loss of YME1L1 accelerates OMA1-dependent L-OPA1 cleavage

  • This functional interaction regulates mitochondrial fusion and fission balance

• Complementary substrate specificities:

  • YME1L1 (i-AAA protease): Degrades proteins in the inner membrane with domains exposed to the intermembrane space

  • AFG3L2/SPG7 (m-AAA protease): Processes proteins with domains exposed to the matrix

  • OMA1 and PARL: Process specific substrates in the inner membrane

  • This division of labor ensures comprehensive mitochondrial protein quality control

• Compensatory mechanisms:

  • In the absence of YME1L1, other proteases may partially compensate for some functions

  • Studies of double protease knockouts have revealed synthetic interactions and shared responsibilities

Understanding these interactions is crucial for comprehending the full landscape of mitochondrial proteostasis and the consequences of protease dysfunction in disease states.

What emerging technologies can advance YME1L1 research?

Several cutting-edge technologies hold promise for deepening our understanding of YME1L1 biology:

• Cryo-electron microscopy (cryo-EM):

  • Determination of high-resolution structures of YME1L1 complexes

  • Visualization of substrate engagement and processing mechanisms

  • Structural insights into disease-causing mutations

• Proximity labeling proteomics:

  • APEX2 or BioID fusion to YME1L1 to identify proximal proteins in vivo

  • Mapping the dynamic YME1L1 interactome under various cellular conditions

  • Identification of transient substrates or regulatory partners

• Advanced genome editing:

  • Base editing or prime editing for precise introduction of disease-associated mutations

  • Inducible degradation systems for temporal control of YME1L1 function

  • Mitochondrially targeted CRISPR systems for organelle-specific manipulations

• Single-cell approaches:

  • Single-cell proteomics to analyze cell-to-cell variation in YME1L1 function

  • Live-cell sensors for monitoring YME1L1 activity in real-time

  • Correlative light and electron microscopy to link functional and structural information

• Computational methods:

  • Machine learning approaches to predict YME1L1 substrates

  • Molecular dynamics simulations of substrate processing

  • Systems biology modeling of the mitochondrial protease network

These emerging technologies will enable researchers to address fundamental questions about YME1L1 function with unprecedented precision and detail, potentially revealing new therapeutic opportunities for mitochondrial diseases and cancer.