Recombinant Saccharomyces cerevisiae Protein translocation protein SEC63 (SEC63)

What is SEC63 and what is its primary function?

SEC63 encodes an integral membrane protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae. It is a 73-kDa polypeptide that localizes to the nuclear envelope-ER network . Its primary function involves facilitating the transport of polypeptides across or into the ER membrane through the Sec61 translocon, which forms an aqueous pore allowing polypeptides to be transferred across or integrated into membranes .

For researchers investigating SEC63 function, methodological approaches include:

• Complementation studies in temperature-sensitive SEC63 mutant strains

• Monitoring translocation efficiency of reporter proteins (e.g., preprolactin, invertase)

• In vitro translocation assays using purified microsomes

• Genetic interaction screens to identify functional partners

What is the structure and topology of SEC63 in the ER membrane?

SEC63 is an integral membrane protein with a well-defined topology that has been characterized through various experimental approaches. In S. cerevisiae, SEC63p contains three transmembrane domains with a crucial lumenal loop containing a DnaJ domain .

The topology has been determined using SEC63-SUC2 (invertase) fusion genes. These experiments revealed that the carboxyl terminus faces the cytosol, as evidenced by unglycosylated hybrid proteins. Conversely, invertase fusion to a loop flanked by two transmembrane domains produces an extensively glycosylated hybrid protein, indicating that this loop faces the ER lumen .

This lumenal loop contains the DnaJ domain, which is homologous to the amino terminus of Escherichia coli heat shock protein DnaJ. The domain is critical for recruiting luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus .

Methods for studying SEC63 topology include:

• Creating fusion proteins with glycosylation-sensitive reporters

• Protease protection assays with isolated microsomes

• Site-specific labeling of cysteine residues

• Immunofluorescence microscopy with domain-specific antibodies

• Cell fractionation combined with biochemical analyses

How does SEC63 interact with other components of the translocation machinery?

SEC63 engages in multiple protein-protein interactions essential for translocation:

• Interaction with Sec61 complex: SEC63 associates with Sec61p (the main channel component), a 31.5-kDa glycoprotein, and a 23-kDa protein, forming part of the polypeptide translocation apparatus .

• Interaction with BiP/Kar2p: The DnaJ domain in SEC63p interacts with the ER luminal BiP/Kar2p (an Hsp70 chaperone), by analogy to the interaction between DnaJ and DnaK proteins in E. coli . This interaction is functionally significant, as both Sec63p and Kar2p are required for efficient protein translocation .

• Complex formation: In yeast, SEC63 is part of the Sec62/Sec63 complex involved in post-translational translocation . Interestingly, in mammalian cells, overexpressed Sec63 exerts its regulatory activity independent of its Sec62-interacting motif, suggesting species-specific differences in complex formation .

Methodological approaches to study these interactions include:

• Co-immunoprecipitation with specific antibodies

• Chemical cross-linking followed by mass spectrometry

• Yeast two-hybrid or split-ubiquitin assays

• Blue native PAGE to analyze intact complexes

• Surface plasmon resonance to measure binding kinetics

• FRET-based interaction assays in living cells

What experimental systems are typically used to study SEC63 function?

Several experimental systems are employed to investigate SEC63 function:

• Yeast genetic systems:

  • Temperature-sensitive mutants (e.g., sec63-101)

  • Suppressor screens to identify genetic interactors (e.g., HSS1)

  • Deletion strains with plasmid-based complementation

• Mammalian cell culture systems:

  • Overexpression and knockdown studies to analyze effects on various ER cargoes

  • Stable cell lines with inducible SEC63 expression

  • CRISPR/Cas9-mediated genome editing

• In vitro systems:

  • Reconstituted proteoliposomes with purified components

  • Rough microsomes isolated from cells with manipulated SEC63 levels

  • Cell-free translation systems coupled with translocation

• Reporter assays:

  • Enzymatic reporters (invertase, alkaline phosphatase)

  • Fluorescent protein-based translocation reporters

  • Glycosylation-dependent mobility shift assays

• Structural biology approaches:

  • Cryo-electron microscopy of translocon complexes

  • X-ray crystallography of individual domains

  • NMR studies of soluble domains

When designing experiments, researchers should consider:

• The specific translocation pathway being studied (co- vs. post-translational)

• The nature of substrate proteins (soluble vs. membrane proteins)

• Species-specific differences between yeast and mammalian systems

• Potential compensatory mechanisms when manipulating SEC63 levels

What are the critical domains in SEC63 protein and their functions?

SEC63 contains several critical domains essential for its function:

• DnaJ domain: Located in the ER-luminal loop between transmembrane domains, this domain is homologous to bacterial DnaJ heat shock proteins. It interacts with BiP/Kar2p (an Hsp70 chaperone) to facilitate translocation . Mutations in two highly conserved positions of this domain inactivate SEC63p activity .

• Transmembrane domains: SEC63 contains three transmembrane segments that anchor it in the ER membrane and establish its topology . These domains may also contribute to interactions with other translocon components.

• Cytosolic C-terminus: The carboxyl terminus faces the cytosol and contains functionally important regions, as short deletions in this region can inactivate SEC63p activity .

• Sec62-interacting region: In mammalian cells, SEC63 contains a region that allows interaction with SEC62, although studies suggest that overexpressed SEC63 can function independently of this interaction in certain contexts .

Methods for studying domain functions include:

• Site-directed mutagenesis of conserved residues

• Domain deletion and swapping experiments

• Expression of isolated domains to identify minimal functional units

• Chimeric constructs between yeast and mammalian SEC63

• Complementation assays with domain mutants in SEC63-deficient cells

How do mutations in the DnaJ domain affect SEC63 function and what methodologies can best detect these effects?

Mutations in the DnaJ domain critically impact SEC63 function through several mechanisms:

• BiP/Kar2p interaction: A J domain-specific mutation that weakens interaction with BiP reduces the regulatory capacity of excess SEC63, confirming BiP involvement in SEC63 function . Mutations in highly conserved positions completely inactivate SEC63p activity .

• Complex formation independence: Interestingly, a nonfunctional DnaJ domain mutant allele does not interfere with the formation of the SEC63p/Sec61p/gp31.5/p23 complex, indicating that complex assembly and functional activity are separable processes .

• Substrate-specific effects: DnaJ domain mutations may affect different substrate proteins to varying degrees, particularly distinguishing between soluble proteins and multi-spanning membrane proteins.

Methodologies to detect and characterize these effects include:

• In vitro biochemical assays:

  • ATPase stimulation assays measuring BiP/Kar2p activity

  • Surface plasmon resonance to quantify binding affinities

  • Isothermal titration calorimetry for thermodynamic parameters

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Translocation efficiency measurements:

  • Pulse-chase experiments with radiolabeled precursors

  • Protease protection assays to assess translocation completeness

  • Glycosylation site accessibility as markers of proper translocation

• Structural analysis:

  • NMR spectroscopy of isolated DnaJ domains

  • Crystallography of DnaJ-BiP complexes

  • Molecular dynamics simulations to predict mutational effects

• Systems-level approaches:

  • Proteomics to identify globally affected substrates

  • Ribosome profiling to detect translational pausing

  • Genetic interaction mapping to identify functional networks

For optimal experimental design, researchers should include multiple substrate classes and combine in vivo and in vitro approaches to comprehensively characterize DnaJ domain mutant phenotypes.

What are the differences between yeast and mammalian SEC63 functions, and how can these be experimentally investigated?

Yeast and mammalian SEC63 show both conserved and divergent functions:

Conserved elements:

• Both are integral ER membrane proteins with J-domains that interact with BiP/Kar2p

• Both participate in protein translocation processes

• Both contain DnaJ domains essential for BiP/Kar2p interaction

Divergent elements:

• Mammalian SEC63 contains ribosome-binding sites at its N-terminus absent in yeast SEC63

• Mammalian SEC63 appears to have substrate-selective regulatory functions, particularly affecting multi-spanning membrane proteins

• While yeast SEC63 typically works closely with SEC62, mammalian SEC63 can function independently of its SEC62-interacting motif

• The requirement for additional components (Sec71p, Sec72p) differs between species

Methodological approaches to investigate these differences:

• Complementation studies:

  • Express mammalian SEC63 in yeast sec63 mutants

  • Test whether yeast SEC63 can rescue defects in mammalian cells with SEC63 knockdown

• Substrate specificity analysis:

  • Compare translocation efficiency of identical substrates in both systems

  • Create substrate libraries with varying signal sequences and transmembrane domains

  • Use quantitative proteomics to identify differentially affected proteins

• Structure-function analysis:

  • Domain swapping between yeast and mammalian SEC63

  • Mutational scanning of conserved and non-conserved regions

  • Biochemical characterization of species-specific interactions

• Ribosome interaction studies:

  • Ribosome binding assays with mammalian SEC63

  • Analysis of ribosome-nascent chain complex interactions

  • Cryo-EM structures of species-specific translocon-ribosome complexes

• Comparative interactome mapping:

  • Mass spectrometry after immunoprecipitation from both systems

  • Proximity labeling (BioID, APEX) to identify context-specific partners

  • Cross-linking mass spectrometry to map interaction surfaces

These approaches would help delineate the evolutionary conservation and divergence of SEC63 function, potentially revealing specialized adaptations in different organisms.

How can researchers distinguish between the roles of SEC63 in SRP-dependent versus SRP-independent translocation?

Distinguishing SEC63's roles in different translocation pathways requires strategic experimental approaches:

• Pathway-specific substrate selection:

  • SRP-dependent pathway: Use substrates with highly hydrophobic signal sequences or transmembrane domains

  • SRP-independent pathway: Use substrates with less hydrophobic signal sequences or short secretory proteins

• Genetic approaches:

  • Create conditional double mutants affecting both SEC63 and SRP components

  • Analyze synthetic genetic interactions between SEC63 and pathway-specific factors

  • Use suppressor screens to identify pathway-specific functional relationships

• In vitro reconstitution strategies:

  • Reconstitute translocation systems with defined components

  • Selectively deplete or inhibit SRP to isolate SRP-independent functions

  • Compare requirements for ATP, GTP, and different chaperones

• Biochemical separation of pathways:

  • Isolate ribosome-nascent chain complexes with or without SRP

  • Use chemical crosslinking to capture pathway-specific intermediates

  • Perform sequential immunodepletion of components from translation lysates

• Real-time imaging approaches:

  • Develop FRET reporters to monitor SEC63 interactions during translocation

  • Use single-molecule fluorescence to track individual translocation events

  • Implement pulse-chase imaging of fluorescent substrate proteins

• Quantitative comparative analysis:

  • Measure the kinetics of each pathway with and without functional SEC63

  • Determine the relative contribution of SEC63 to each pathway's efficiency

  • Assess how SEC63 mutations differentially affect each pathway

Research has demonstrated that SEC63 and Kar2p are required for the SRP-dependent targeting pathway in vivo , contradicting earlier models that suggested their involvement only in SRP-independent translocation. This finding highlights the need for careful experimental design that can distinguish pathway-specific functions.

What advanced techniques can be used to study the dynamic interactions between SEC63 and other translocon components?

Understanding the dynamic interactions of SEC63 requires sophisticated methodological approaches:

• Structural biology techniques:

  • Cryo-electron microscopy of intact translocon complexes in different functional states

  • Time-resolved X-ray crystallography of individual domains or subcomplexes

  • Nuclear magnetic resonance (NMR) of soluble domains and their interactions

  • Integrative structural modeling combining multiple data sources

• Advanced fluorescence techniques:

  • Single-molecule FRET to measure distances between components

  • Fluorescence correlation spectroscopy (FCS) to analyze binding kinetics

  • Super-resolution microscopy (STORM, PALM) to visualize translocon organization

  • Fluorescence recovery after photobleaching (FRAP) to measure dynamics

• Proteomics approaches:

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

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify conformational changes

  • Thermal proteome profiling to detect stability changes upon complex formation

  • Quantitative interaction proteomics under various conditions

• Proximity labeling methods:

  • BioID or TurboID fused to SEC63 to identify neighboring proteins

  • APEX2-mediated biotinylation for millisecond-scale proximity mapping

  • Split-enzyme complementation to detect specific interactions

• Computational approaches:

  • Molecular dynamics simulations of translocon complexes

  • Coevolution analysis to predict interaction surfaces

  • AlphaFold2-based modeling of complex architectures

• Real-time functional assays:

  • Optical tweezers to measure forces during translocation

  • Nanopore recording of single translocation events

  • Tethered ribosome systems to monitor nascent chain interactions

• In-cell structural techniques:

  • FRET sensors to monitor conformational changes in living cells

  • Genetic code expansion for site-specific photo-crosslinking

  • Intramolecular distance measurements using lanthanide resonance energy transfer

For optimal experimental design, researchers should combine complementary approaches and develop assays that can differentiate between stable and transient interactions in the dynamic translocation process.

How can researchers quantitatively assess the impact of SEC63 manipulations on specific substrate proteins?

Quantitative assessment of SEC63's impact on substrate proteins requires precise methodological approaches:

• Steady-state protein level analysis:

  • Quantitative western blotting with fluorescent secondary antibodies

  • ELISA for secreted proteins

  • Flow cytometry for cell-surface reporters

  • Targeted mass spectrometry (SRM/MRM/PRM) for absolute quantification

• Protein synthesis and degradation kinetics:

  • Pulse-chase experiments with radioisotope or stable isotope labeling

  • Cycloheximide chase assays to monitor turnover rates

  • Ribosome profiling to measure translation efficiency

  • Ubiquitination assays to assess degradation targeting

• Translocation efficiency measurements:

  • Protease protection assays with quantitative readouts

  • Glycosylation site accessibility as translocation markers

  • Signal sequence cleavage efficiency

  • Compartment-specific labeling techniques

• High-throughput screening approaches:

  • Reporter constructs with luminescent or fluorescent readouts

  • Flow cytometry-based sorting of cellular populations

  • Automated microscopy with image analysis

  • Pooled CRISPR screens with SEC63 variants

• Single-cell analysis:

  • Time-lapse fluorescence microscopy

  • Single-cell RNA-seq combined with proteomics

  • Microfluidic approaches to monitor individual cell responses

  • Cell-to-cell variability assessment

Research has shown that overexpression of Sec63 reduces steady-state levels of multi-spanning membrane proteins while soluble and single-spanning reporters remain unaffected. Conversely, Sec63 knockdown increases polytopic protein levels . These findings highlight the importance of testing multiple substrate classes when assessing SEC63 function.

The following table summarizes key approaches for different substrate types:

What are the challenges in reconstituting SEC63 function in vitro and how can these be addressed?

Reconstituting SEC63 function in vitro presents several technical challenges:

• Membrane protein purification challenges:

  • SEC63 contains multiple transmembrane domains making purification difficult

  • Detergent selection is critical to maintain native conformation

  • Protein may denature or aggregate during purification

• Complex reconstitution requirements:

  • SEC63 functions as part of larger protein complexes

  • Multiple components must be co-purified or added sequentially

  • Stoichiometry must be carefully controlled

• Maintaining native topology:

  • Ensuring correct orientation in artificial membranes

  • Verifying proper membrane insertion

  • Confirming luminal vs. cytosolic domain exposure

• Functional assay limitations:

  • Detecting successful translocation events

  • Distinguishing partial from complete translocation

  • Recreating the energy requirements (ATP/GTP)

Methodological solutions include:

• Advanced purification strategies:

  • Mild detergent selection (DDM, LMNG)

  • Lipid nanodiscs or amphipols for membrane mimetics

  • Styrene maleic acid lipid particles (SMALPs) for native membrane extraction

  • Co-expression systems for intact complexes

• Membrane reconstitution approaches:

  • Controlled proteoliposome formation through detergent dialysis

  • Giant unilamellar vesicles (GUVs) for microscopy-based assays

  • Planar lipid bilayers for electrical recordings

  • Defined lipid compositions matching ER membranes

• Functionality verification:

  • BiP/Kar2p ATPase stimulation assays for J-domain activity

  • Protease protection assays for translocation

  • Site-specific labeling to confirm topology

  • Crosslinking to verify protein-protein interactions

• Alternative systems:

  • Semi-permeabilized cells with manipulated SEC63 levels

  • Rough microsomes with immunodepleted/reconstituted components

  • Cell-free translation-translocation systems

  • Hybrid systems combining purified components with native membranes

These methodological approaches can help overcome the challenges of reconstituting SEC63 function in vitro, enabling detailed mechanistic studies of its role in protein translocation.

How does SEC63 contribute to quality control of membrane proteins, and what methodologies can be used to study this function?

Evidence suggests SEC63 plays a regulatory role in membrane protein quality control:

• Substrate-specific regulation: Overexpression of SEC63 reduces steady-state levels of multi-spanning membrane proteins while soluble and single-spanning proteins remain unaffected. Conversely, SEC63 knockdown increases polytopic protein levels .

• BiP-dependent mechanism: A J domain-specific mutation of SEC63 that weakens BiP interaction reduces its regulatory capacity, suggesting BiP involvement in this quality control function .

• Selective action: The specificity for polytopic membrane proteins suggests a role in monitoring complex membrane protein integration or folding .

Methodological approaches to study this function include:

• Membrane protein fate tracking:

  • Pulse-chase analysis with detection of different maturation states

  • Ubiquitination profiling of membrane protein substrates

  • Subcellular fractionation to track protein localization

  • Co-immunoprecipitation with quality control machinery components

• ERAD pathway analysis:

  • Comparison of SEC63 effects with and without proteasome inhibitors

  • Assessment of interactions with ERAD components

  • Measurement of retrotranslocation efficiency

  • Analysis of SEC63 requirements for different ERAD substrates

• Folding state assessment:

  • Limited proteolysis to detect conformational differences

  • Conformation-specific antibodies to distinguish folding states

  • Thermal stability assays for membrane proteins

  • FRET-based folding sensors incorporated into substrate proteins

• BiP interaction studies:

  • Co-immunoprecipitation under native conditions

  • Use of BiP mutants (ATP-binding, hydrolysis-deficient)

  • Client release assays with nucleotide exchange factors

  • Competition experiments with other J-domain proteins

• High-resolution imaging:

  • Visualization of membrane protein aggregation

  • Co-localization with quality control compartments

  • Live-cell tracking of membrane protein trafficking

  • Super-resolution microscopy of quality control sites

The following table summarizes the differential effects of SEC63 manipulations on various substrate types: