Recombinant Drosophila yakuba DDRGK domain-containing protein 1 (GE25716)

What is the primary structure of Drosophila yakuba DDRGK domain-containing protein 1?

The Drosophila yakuba DDRGK domain-containing protein 1 (GE25716) is a 312 amino acid protein with UniProt accession B4PQC4. The complete amino acid sequence has been determined and is available in the Autoinhibited Protein Database (AiPD) . The protein contains a characteristic DDRGK domain and is predicted to have potential autoinhibitory elements via cis-regPred analysis . The primary sequence includes a putative signal peptide at the N-terminus that likely contributes to its endoplasmic reticulum anchoring, similar to homologous proteins in other species .

The complete amino acid sequence is presented below:

What structural predictions are available for Drosophila yakuba DDRGK1?

The structure of Drosophila yakuba DDRGK1 has been predicted using computational methods, with a model available in AlphaFoldDB (entry AF-B4PQC4-F1) . The AiPD database indicates that this protein may contain autoinhibitory domains (AIDs) identified through cis-regPred analysis . While detailed structural information specific to D. yakuba DDRGK1 is limited, comparisons with homologous proteins suggest it contains:

• A conserved DDRGK domain crucial for its role in the ufmylation system

• A proteasome component (PCI) domain implicated in protein interactions

• A signal peptide at the N-terminus for endoplasmic reticulum anchoring

No crystal structure of the D. yakuba DDRGK1 has been reported yet, leaving important questions about its three-dimensional conformation and the structural basis of its autoinhibitory mechanism unresolved.

How does DDRGK1 function in the ufmylation pathway?

DDRGK1 is a critical component of the ufmylation system, a ubiquitin-like modification pathway. Based on studies of DDRGK1 homologs, the protein functions as follows:

• DDRGK1 serves as a major target for ufmylation by Ufm1 (ubiquitin-fold modifier 1)

• The ufmylation process involves:

  • Activation of Ufm1 by the E1 enzyme (Uba5)

  • Transfer to the E2 enzyme (Ufc1)

  • Conjugation to target proteins via the E3 ligase (Ufl1)

• DDRGK1 forms a complex with other ufmylation components including Ufl1, C53/LZAP (Cdk5rap3), and Ufm1 at the endoplasmic reticulum

This system is essential for maintaining endoplasmic reticulum homeostasis, with DDRGK1 playing a central role in this process. Disruption of DDRGK1 function can lead to extensive ER stress, highlighting its importance in cellular physiology .

What are optimal expression systems for recombinant Drosophila yakuba DDRGK1?

When expressing recombinant D. yakuba DDRGK1, researchers should consider several expression systems, each with distinct advantages:

For structural studies requiring correctly folded protein, insect cell or Drosophila S2 cell expression is preferred. Consider the addition of N-terminal signal sequences to ensure proper targeting to the endoplasmic reticulum, reflecting the native localization of DDRGK1. For functional assays examining ufmylation, co-expression with other pathway components (Ufm1, Uba5, Ufc1, Ufl1) may be necessary.

What purification challenges are specific to DDRGK1?

Purification of D. yakuba DDRGK1 presents several challenges researchers should address:

• Membrane association: The N-terminal signal peptide suggests DDRGK1 is associated with the ER membrane , requiring careful solubilization strategies:

  • Test mild detergents (DDM, LMNG, digitonin)

  • Consider using truncation constructs lacking the N-terminal region

• Potential autoinhibitory domains: The presence of autoinhibitory domains may affect protein stability and homogeneity:

  • Design constructs that stabilize specific conformational states

  • Consider co-expression with binding partners that might relieve autoinhibition

• Protein stability considerations:

  • Include protease inhibitors throughout purification

  • Test various buffer compositions (pH 6.5-8.0, salt concentrations 150-500 mM)

  • Consider stabilizing additives (5-10% glycerol, 1 mM DTT)

• Tag selection and removal:

  • Test both N- and C-terminal tags to identify optimal positioning

  • Include TEV or PreScission protease cleavage sites for tag removal

  • Validate that tag removal doesn't impact protein stability

Assess protein quality using multiple techniques including size exclusion chromatography, dynamic light scattering, and thermal shift assays to ensure homogeneity and stability before proceeding to functional studies.

What techniques are recommended for studying DDRGK1 protein interactions?

To comprehensively characterize D. yakuba DDRGK1 protein interactions, a multi-pronged methodological approach is recommended:

• Co-immunoprecipitation and pull-down assays:

  • Use recombinant tagged DDRGK1 as bait protein

  • Apply to lysates from D. yakuba tissues

  • Identify interacting partners via mass spectrometry

  • Based on mammalian studies, focus on potential interactions with v-ATPase subunits like Atp6v0d1

• Proximity-based labeling techniques:

  • BioID or TurboID fusion with DDRGK1

  • APEX2-based proximity labeling

  • These methods are particularly valuable for capturing transient or weak interactions

• Biophysical interaction characterization:

  • Surface plasmon resonance (SPR) or biolayer interferometry (BLI)

  • Isothermal titration calorimetry (ITC)

  • Microscale thermophoresis (MST)

  • These techniques provide quantitative binding parameters (Kd, kon, koff)

• Structural studies of complexes:

  • Crosslinking mass spectrometry (XL-MS) to map interaction interfaces

  • Cryo-electron microscopy for larger complexes

  • X-ray crystallography for high-resolution structural details

Studies in other systems have shown that DDRGK1 interacts with Atp6v0d1 and affects proteasome-mediated degradation of v-ATPase subunits , suggesting these would be priority targets for interaction studies in D. yakuba.

How does DDRGK1 regulate autophagy in cellular systems?

Based on studies of DDRGK1 homologs, this protein has a dual role in autophagy regulation:

• Autophagy induction effects:

  • DDRGK1 influences the mTOR signaling pathway

  • DDRGK1 deficiency leads to mTOR inactivation and AMPK activation, promoting autophagy initiation

  • This results in increased autophagosome formation

• Autophagy degradation effects:

  • DDRGK1 is required for proper autophagosome-lysosome fusion

  • It maintains normal lysosomal function and proper expression of lysosomal enzymes

  • Its absence impairs autophagic flux, blocking the degradation phase

• Lysosomal function regulation:

  • DDRGK1 colocalizes with lysosomal marker LAMP2

  • It interacts with v-ATPase subunits like Atp6v0d1

  • It modulates lysosomal pH by regulating v-ATPase activity

  • DDRGK1 deficiency affects Cathepsin D (CTSD) expression

The dual role explains why DDRGK1 deficiency leads to abnormal accumulation of autophagosomes—increased formation coupled with decreased clearance—ultimately resulting in cellular dysfunction and increased apoptosis .

What experimental approaches can detect alterations in autophagic flux mediated by DDRGK1?

To investigate DDRGK1's impact on autophagic flux, researchers should employ multiple complementary techniques:

• Autophagosome formation markers:

  • LC3-II western blotting with and without lysosomal inhibitors

  • GFP-LC3 puncta formation via fluorescence microscopy

  • Transmission electron microscopy to visualize autophagosome accumulation

• Autophagic flux assessment:

  • Tandem mRFP-GFP-LC3 reporter assay to distinguish autophagosomes from autolysosomes

  • Long-lived protein degradation assays using radiolabeled amino acids

  • p62/SQSTM1 accumulation as indicator of impaired autophagic degradation

• Lysosomal function evaluation:

  • LysoTracker staining to assess lysosomal acidification (critical since DDRGK1 affects lysosomal pH)

  • Activity assays for lysosomal enzymes including Cathepsin D

  • Analysis of v-ATPase subunit levels (particularly Atp6v0d1 and Atp6v1a)

• Signaling pathway analysis:

  • Phosphorylation status of mTOR, S6K, and AMPK

  • ULK1 phosphorylation and activity assessment

  • TFEB nuclear translocation as indicator of lysosomal biogenesis

• Genetic complementation:

  • DDRGK1 knockout/knockdown with rescue experiments

  • Domain-specific mutants to identify regions critical for autophagy regulation

These approaches should be performed under various conditions, including basal, starvation-induced, and chemically-induced autophagy, to comprehensively characterize DDRGK1's role.

How does DDRGK1 deficiency impact cellular physiology in model systems?

DDRGK1 deficiency produces multiple cellular and physiological consequences:

• Endoplasmic reticulum stress:

  • Induction of the unfolded protein response (UPR)

  • Activation of ER stress sensors (IRE1α, PERK, ATF6)

  • Potential disruption of protein secretion and processing

• Autophagy dysregulation:

  • Abnormal accumulation of autophagosomes due to:

    • Enhanced autophagy induction via mTOR inhibition

    • Impaired autophagosome-lysosome fusion

  • Compromised clearance of cellular waste and damaged organelles

• Lysosomal dysfunction:

  • Altered lysosomal pH (observed as increased LysoTracker staining)

  • Reduced Cathepsin D expression

  • Abnormal accumulation of v-ATPase subunits (Atp6v0d1 and Atp6v1a)

• Cellular stress and death:

  • Increased apoptosis due to prolonged ER stress and impaired autophagy

  • Potential accumulation of protein aggregates

  • Mitochondrial dysfunction secondary to impaired mitophagy

• Developmental and physiological consequences:

  • While not specifically studied in D. yakuba, DDRGK1 deficiency in other systems leads to:

    • Hematopoietic dysfunction

    • Impaired intestinal homeostasis

    • Compromised plasma cell development

These findings illustrate DDRGK1's essential role in maintaining cellular homeostasis through its involvement in ufmylation, ER function, and autophagy-lysosome processes.

How conserved is DDRGK1 across Drosophila species and broader phylogeny?

The DDRGK domain-containing protein 1 shows interesting patterns of conservation across species:

• Conservation within Drosophila:

  • D. yakuba DDRGK1 (GE25716) shows high sequence conservation with orthologs in other Drosophila species

  • The DDRGK domain itself is highly conserved across the Drosophila genus

  • Comparative genomics between D. yakuba and other Drosophila species provides insights into functional elements

• Conservation across broader phylogeny:

  • The DDRGK domain is evolutionarily conserved from insects to mammals

  • Key functional regions show higher conservation than non-functional regions

  • The N-terminal signal peptide and DDRGK domain are particularly well conserved

• Functional implications of conservation:

  • High conservation suggests fundamental cellular roles

  • DDRGK1's involvement in the ufmylation system and ER homeostasis appears to be an ancient and conserved function

  • Similar roles in autophagy regulation likely exist across diverse species

• Divergent features:

  • Species-specific variations may reflect adaptation to different physiological requirements

  • Regulatory elements controlling expression show greater divergence than coding sequences

The conservation of DDRGK1 across species supports its critical role in fundamental cellular processes and makes Drosophila yakuba a valuable model for studying its function.

What can comparative studies between D. yakuba DDRGK1 and mammalian DDRGK1 reveal?

Comparative analysis between D. yakuba and mammalian DDRGK1 proteins yields valuable insights:

• Structural similarities and differences:

  • Both contain conserved DDRGK domains essential for ufmylation

  • Both possess N-terminal signal peptides for ER localization

  • Potential differences in autoinhibitory mechanisms and regulatory elements

• Functional conservation:

  • Both participate in the ufmylation pathway

  • Both play roles in maintaining ER homeostasis

  • Mammalian DDRGK1 regulates autophagy and lysosomal function , suggesting similar functions may exist in D. yakuba

• Experimental advantages of cross-species studies:

  • Drosophila provides a genetically tractable system for studying DDRGK1 function

  • Mammalian cell studies offer translational relevance

  • Complementation experiments can test functional conservation

• Key differences to investigate:

  • Mammalian DDRGK1 interacts with v-ATPase subunits and regulates their proteasomal degradation

  • Mammalian DDRGK1 affects Cathepsin D expression and lysosomal acidification

  • Whether these specific mechanisms are conserved in D. yakuba remains to be determined

• Evolutionary implications:

  • Conserved functions likely represent ancient cellular mechanisms

  • Divergent functions may reveal lineage-specific adaptations

  • Understanding both conserved and divergent aspects provides evolutionary context

This comparative approach can leverage findings across species to accelerate understanding of DDRGK1 function in both systems.

How can CRISPR-Cas9 genome editing be optimized for studying D. yakuba DDRGK1?

Optimizing CRISPR-Cas9 genome editing for D. yakuba DDRGK1 research requires specific methodological considerations:

• Guide RNA design strategy:

  • Select target sites with minimal off-target potential

  • Design multiple gRNAs targeting different exons of DDRGK1

  • Prioritize targeting conserved functional domains like the DDRGK domain

  • Consider the GC content of target sites (40-60% optimal)

• Delivery methods for D. yakuba:

  • Embryo microinjection of Cas9 protein and gRNA complexes

  • Integration of Cas9 and gRNA expression constructs via P-element or PhiC31 systems

  • Optimize injection timing based on D. yakuba embryonic development

• Modification strategies:

  • Complete gene knockout through frameshift mutations

  • Domain-specific mutations to assess functions of individual regions

  • Knock-in of tags (GFP, FLAG) for localization and interaction studies

  • Conditional alleles using FLP/FRT or Gal4/UAS systems

• Validation methods:

  • Genomic PCR and sequencing to confirm edits

  • RT-PCR and western blot to assess transcript and protein levels

  • Functional assays based on expected phenotypes:

    • ER stress markers

    • Autophagy flux measurements

    • Lysosomal function assessment

• Phenotypic analysis:

  • Developmental timing and morphology

  • Cellular stress responses

  • Lifespan and stress resistance

  • Tissue-specific effects in systems known to be sensitive to DDRGK1 loss

By implementing these optimized approaches, researchers can generate valuable genetic tools for dissecting DDRGK1 function in D. yakuba.

How might post-translational modifications regulate D. yakuba DDRGK1?

Post-translational modifications (PTMs) likely play crucial roles in regulating D. yakuba DDRGK1 function:

• Ufmylation:

  • DDRGK1 itself is a primary target of ufmylation

  • Potential ufmylation sites should be identified through mass spectrometry

  • Functional consequences may include altered stability, localization, or protein interactions

  • Site-directed mutagenesis of ufmylation sites can reveal their functional importance

• Phosphorylation:

  • Predictive algorithms can identify potential phosphorylation sites

  • Phosphorylation may regulate:

    • Protein-protein interactions

    • Subcellular localization

    • Relief of autoinhibition

  • Kinase inhibitor studies and phospho-specific antibodies can probe regulation

• Other potential modifications:

  • Ubiquitination: May regulate DDRGK1 stability and turnover

  • Glycosylation: Potential role in ER localization and folding

  • Palmitoylation: Could affect membrane association

• Methodological approaches:

  • Mass spectrometry-based PTM mapping

  • Site-directed mutagenesis of modified residues

  • Pharmacological manipulation of modification pathways

  • Generation of modification-specific antibodies

• Regulatory implications:

  • PTMs likely create a dynamic regulatory network

  • Different cellular conditions may induce specific modification patterns

  • Cross-talk between different modification types may create complex regulatory logic

Understanding this PTM network will be essential for deciphering how DDRGK1 function is regulated in response to changing cellular conditions.

How can contradictions between structural predictions and functional observations of DDRGK1 be resolved?

Resolving contradictions between structural predictions and functional observations requires a multi-faceted approach:

• Limitations of current structural data:

  • The AlphaFoldDB prediction (AF-B4PQC4-F1) provides a computational model

  • Predicted autoinhibitory elements from cis-regPred require experimental validation

  • Dynamic conformational changes may not be captured in static models

• Experimental structure determination:

  • X-ray crystallography of full-length protein or functional domains

  • Cryo-electron microscopy, particularly for complexes with interaction partners

  • Nuclear magnetic resonance (NMR) for dynamic regions and smaller domains

  • Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

• Structure-function correlation approaches:

  • Site-directed mutagenesis guided by structural predictions

  • Functional assays to test activity of mutant variants:

    • Ufmylation assays

    • Autophagy regulation

    • Interaction with v-ATPase subunits

  • Truncation constructs to isolate functional domains

• Computational refinement:

  • Molecular dynamics simulations to model protein flexibility

  • Integration of experimental constraints into structural models

  • Docking studies with interaction partners

• Comparative analysis:

  • Structural comparison with better-characterized homologs

  • Conservation analysis to identify functionally important residues

  • Evolutionary covariance analysis to predict interacting regions

This integrated approach can bridge the gap between structural predictions and functional observations, leading to a more comprehensive understanding of DDRGK1 structure-function relationships.

What are the key unanswered questions about D. yakuba DDRGK1?

Despite growing understanding of DDRGK domain-containing protein 1, several critical questions remain:

• Structural mechanisms:

  • How do the predicted autoinhibitory elements regulate DDRGK1 function?

  • What is the atomic-level structure of D. yakuba DDRGK1, particularly in different functional states?

  • How does ufmylation alter DDRGK1 structure and function?

• Functional mechanisms:

  • Is the dual role in autophagy regulation observed in mammalian systems conserved in D. yakuba?

  • How does DDRGK1 precisely regulate v-ATPase subunits and lysosomal function in D. yakuba?

  • What is the comprehensive interactome of DDRGK1 in D. yakuba cells?

• Physiological relevance:

  • What are the developmental and tissue-specific functions of DDRGK1 in D. yakuba?

  • How does DDRGK1 contribute to stress responses and longevity?

  • Are there species-specific adaptations in DDRGK1 function between different Drosophila species?

• Evolutionary aspects:

  • How has DDRGK1 function evolved across phylogeny?

  • Which aspects of DDRGK1 function are ancestral versus derived?

  • How do regulatory mechanisms differ between insects and mammals?

Addressing these questions will require integrated approaches combining structural biology, biochemistry, cell biology, genetics, and evolutionary analysis.

What are the most promising directions for future DDRGK1 research?

Future research on D. yakuba DDRGK1 should focus on several promising directions:

• Comprehensive structure-function analysis:

  • Determination of high-resolution structures in different functional states

  • Mapping of interaction interfaces with binding partners

  • Characterization of conformational dynamics and autoinhibitory mechanisms

• Systems-level understanding:

  • Integration of transcriptomics, proteomics, and metabolomics data

  • Network analysis of DDRGK1-dependent pathways

  • Computational modeling of DDRGK1's role in cellular homeostasis

• Physiological roles:

  • Generation of tissue-specific knockout models

  • Analysis of developmental roles and stress responses

  • Investigation of interspecies differences in DDRGK1 function

• Translational implications:

  • Comparison with human DDRGK1 function

  • Potential relevance to understanding disease mechanisms

  • Exploration of DDRGK1 as a therapeutic target

• Advanced methodological approaches:

  • Cryo-electron tomography to visualize DDRGK1 in its native cellular context

  • Live-cell imaging with advanced fluorescent probes

  • Single-molecule studies of DDRGK1 dynamics and interactions