Recombinant Schizosaccharomyces pombe Translocation protein sec63 (sec63)

What is Sec63 and what is its primary function in S. pombe?

Sec63 is an essential transmembrane protein that plays a crucial role in the translocation of secretory polypeptides into the endoplasmic reticulum. In S. pombe, Sec63p functions as part of the translocation machinery that orchestrates the transport and maturation of polypeptides at the ER membrane. The protein works in conjunction with Kar2p/BiP to facilitate both signal recognition particle (SRP)-dependent and SRP-independent translocation pathways .

The Sec63 protein integrates into a larger complex known as the "Sec complex," which comprises the Sec61 trimer (Sec61p, Sss1p, and Sbh1p) plus the tetrameric Sec63 complex composed of two essential proteins (Sec62p and Sec63p) plus two non-essential proteins (Sec71p and Sec72p) . This complex forms the core machinery responsible for protein translocation across the ER membrane in fission yeast.

How is the structure of Sec63 organized?

Sec63 has several distinct structural domains that contribute to its function. Based on cryo-EM structural data, Sec63 contains regions that are missing in the structural models, including the J-domain (86 amino acids long) and 63 amino-acid residues from the C-terminus . The J-domain is particularly important as it facilitates the recruitment and interaction with BiP/Kar2p.

How does Sec63 interact with other components of the translocation machinery?

Sec63 forms specific interactions with several components of the translocation machinery:

• Interaction with Sec61 complex: Sec63 associates with the Sec61 translocon, which forms the central pore through which proteins are translocated. This interaction is important for stabilizing the entire translocation complex .

• Interaction with BiP/Kar2p: The J-domain of Sec63 recruits and activates BiP/Kar2p, an ER lumenal chaperone that provides the driving force for post-translational translocation .

• Interaction with substrate proteins: Sec63 helps recognize specific features in signal peptides of substrate proteins, particularly those with slowly gating signal peptides or translation-disruptive positively charged amino acid clusters .

Molecular dynamics simulations have shown that Sec63 binding affects the conformation of Sec61, suggesting a regulatory role in controlling the opening and closing of the translocation channel .

What are the distinct roles of Sec63 in SRP-dependent versus SRP-independent pathways?

Sec63 plays multiple distinct roles in protein translocation, with differential involvement in SRP-dependent and SRP-independent pathways:

SRP-independent pathway (post-translational):

• Sec63p along with Sec62p, Sec71p, Sec72p, and Kar2p/BiP forms a receptor complex for post-translational translocation .

• It maintains the stability of the heptameric complex essential for this pathway .

• In reconstituted proteoliposomes, the Sec complex plus Kar2p are sufficient for post-translational translocation of pre-pro-α-factor .

SRP-dependent pathway (co-translational):

• Studies with sec63-301 mutant cells demonstrate that Sec63p is substantially defective in SRP-dependent translocation, revealing a role in this pathway as well .

• Unlike the SRP-independent pathway, the SRP-dependent role of Sec63p does not require Sec62p .

• The minimal functional translocon for SRP-dependent translocation comprises the Sec61 complex plus Sec63p/Kar2p .

This dual functionality suggests that Sec63p may support Sec61-channel opening for precursor polypeptides with slowly gating signal peptides, either through direct interaction with the cytosolic amino-terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61α .

How does Sec63 affect the conformation of Sec61 in yeast?

Molecular dynamics simulations have provided insights into how Sec63 influences the Sec61 translocon conformation:

• Conformational stability: Analysis of the root mean square deviation (RMSD) and radius of gyration measurements indicate that after approximately 600 ns of simulation, the conformational relaxation of the unbound Sec61 simulation reaches plateau values, suggesting stabilization .

• Local structural features: Measurements of pore-ring, lateral gate, and plug distances and angles reveal that Sec63 binding affects these local dynamics. The distributions obtained from the last 50 ns of simulations are narrower than those from the last 400 ns, but peak positions remain at similar values in both "free" and "Sec63-bound" states .

• Functional implications: These conformational changes induced by Sec63 likely regulate the opening and closing of the Sec61 channel, thereby controlling the translocation process. The lateral gate of Sec61 is particularly important as it allows signal sequences to enter the channel and hydrophobic segments to exit into the lipid bilayer .

What is the role of the Sec63/BiP complex in cellular stress responses?

The Sec63/BiP complex plays an important role in cellular stress responses, particularly in relation to the unfolded protein response (UPR):

• Interaction with IRE1α: Studies have shown that Sec63 mediates BiP binding to IRE1α, a key sensor of ER stress. This interaction suppresses the formation of higher-order oligomers of IRE1α, leading to proper attenuation of IRE1α RNase activity .

• Translocon-dependent interaction: IRE1α interacts with the Sec61/Sec63 complex, and this interaction is reduced but not abolished in Sec63 knockout cells. This suggests that IRE1α can interact with the translocon independent of Sec63, but preferentially interacts with a Sec61 translocon complex containing Sec63 .

• Regulatory function: Sec63 mutants that poorly interact with the Sec61 translocon also show decreased interaction with endogenous IRE1α, indicating that Sec63's role in stress response regulation is linked to its function in the translocon complex .

What genetic systems are available to study Sec63 function in S. pombe?

Several genetic systems have been developed to study Sec63 function in S. pombe:

• Conditional alleles: Since SEC63 is an essential gene, conditional mutants such as temperature-sensitive alleles (e.g., sec63-301) have been valuable for studying its function. These mutants show defects in specific aspects of translocation while remaining viable under permissive conditions .

• Repressible promoter systems: A strain in which the genomic copy of SEC63 is placed under the control of the repressible MET3 promoter (PMET3-SEC63) allows controlled depletion of Sec63p to study translocation activity in its absence .

• Recombination assays: S. pombe has powerful in vivo genetic assays for studying mitotic recombination, which can be adapted to investigate the role of Sec63 in DNA repair and related processes .

• Rapid transcriptional induction systems: A system based on upregulation of the urg1 promoter allows induction within 30 minutes, similar to the S. cerevisiae GAL induction system. This is much faster than the traditional nmt1 promoter, which requires 14-20 hours for full induction .

How can we identify signal peptide features that determine Sec63 dependency?

Research has identified specific signal peptide features that determine which proteins require Sec63 for efficient translocation:

• Signal peptide characteristics: Proteins requiring Sec63/Sec62 for efficient translocation typically have signal peptides with:

  • Comparatively longer but less hydrophobic regions

  • Lower carboxy-terminal region (C-region) polarity

• Translocation-disruptive elements: The combination of a slowly gating signal peptide and a downstream translocation-disruptive positively charged cluster of amino acid residues appears to be decisive for Sec63 dependency .

• Additional BiP requirement: These signal peptide features often correlate with an additional BiP requirement and sensitivity toward Sec61-channel inhibitors like CAM741 .

• Validation methods: Proteomic approaches followed by independent silencing and western blot experiments can validate Sec63 substrate specificity. For instance, SEC62- and SEC63-targeted siRNAs have been used to confirm specific substrates .

What methodologies are used for structural studies of Sec63?

Multiple complementary approaches have been employed to study the structure and dynamics of Sec63:

• Cryo-EM: This technique has been used to determine the structure of the Sec complex, although with limitations. Long stretches from the N-termini of associated proteins (Sss1 and Sbh1) and structural information of the J-domain of Sec63 are missing in these structures .

• Homology modeling: For missing regions, homology modeling based on related structures (e.g., mammalian Sec61α for yeast Sec61) has been employed. The MODELLER software package has been used for this purpose .

• Molecular dynamics simulations: MD simulations provide insights into the dynamic behavior of Sec63 and its effects on the Sec61 complex. These simulations use atomistic detail with the CHARMM36 force field for lipids and proteins and the TIP3P model for water .

• System preparation for MD simulations:

  • Membrane environment: POPC (palmitoyl-oleoyl-phosphatidylcholine) lipids

  • Physiological conditions: 150 mM KCl, electrically neutral systems

  • System size: 148,218-244,258 atoms including lipids, water molecules, and ions

How does Sec63 contribute to protein quality control in the ER?

Sec63 plays critical roles in protein quality control within the ER:

• Substrate recognition: Sec63 helps identify and process proteins with specific signal peptide features, particularly those with slowly gating signal peptides that might otherwise fail to translocate efficiently .

• BiP recruitment: Through its J-domain, Sec63 recruits and activates BiP/Kar2p, which functions not only in translocation but also in protein folding and quality control within the ER lumen .

• ER stress regulation: The Sec63/BiP complex helps regulate IRE1α activity, a key component of the unfolded protein response. By suppressing higher-order oligomerization of IRE1α, this complex helps ensure proper attenuation of the stress response .

• Translocation efficiency: By ensuring efficient translocation of specific substrate proteins, Sec63 prevents the accumulation of untranslocated or partially translocated proteins that could trigger ER stress and quality control pathways .

What is the significance of Sec63 in mitotic recombination studies?

While Sec63's primary role is in protein translocation, S. pombe assays involving recombination provide important experimental systems:

• Model organism advantages: S. pombe has emerged as a powerful tractable system for studying DNA damage repair, with several powerful in vivo genetic assays developed to study outcomes of mitotic recombination .

• Recombination at repetitive elements: Assays have been devised in S. pombe to study chromosomal recombination at non-tandem repeats, which can cause deletions, inversions, or duplications. These systems can provide insights into cellular processes that may indirectly involve Sec63 .

• Single-strand annealing (SSA): Experiments using S. cerevisiae LEU2 fragments with overlapping regions placed on either side of a functional his3+ gene have shown that SSA is rad52-dependent, which may have implications for understanding cellular repair mechanisms in the context of ER stress and protein translocation defects .

The relationship between Sec63 function in protein translocation and these recombination processes represents an interesting area for future research, potentially linking protein homeostasis with genome stability.