Recombinant Candida albicans Translocation protein SEC62 (SEC62)

What is the basic structure and function of C. albicans SEC62?

SEC62 is a double-spanning membrane protein with large N- and C-terminal soluble domains located in the cytosol. It contains two transmembrane domains (TM1 and TM2), with the N-terminal domain containing clusters of positively charged residues that are critical for interaction with SEC63 and proper function. The C-terminal domain following the second transmembrane region is essential for cell viability . As part of the post-translational translocation machinery, SEC62 facilitates the transport of fully synthesized proteins across the ER membrane by working with other components including SEC63, SEC71, and SEC72 .

How does SEC62 contribute to the process of protein translocation?

SEC62 is a component of the post-translational translocation pathway where fully synthesized proteins are recognized by a specialized channel containing SEC62, SEC63, and in yeast, accessory factors SEC71 and SEC72 . SEC62's transmembrane domains, particularly TM2, help stabilize signal sequences at the lateral gate of the SEC61 translocon. This stabilization is especially important for the recognition of less hydrophobic post-translational signal sequences . Upon substrate engagement, the signal sequence binds to the groove at the lateral gate and is further stabilized by TM2 of SEC62, causing the lateral gate to widen, followed by plug displacement and translocation of the polypeptide chain .

What is the relationship between SEC62 and signal sequence hydrophobicity?

SEC62 shows specificity for moderately hydrophobic signal sequences. Research has demonstrated that proteins with highly hydrophobic signal sequences typically utilize the SRP-dependent co-translational pathway, while those with less hydrophobic signal sequences often rely on the post-translational pathway involving SEC62 . Interestingly, moderately hydrophobic signal sequences appear to require both pathways for optimal targeting and translocation. When SEC62 function is defective, the insertion of moderately hydrophobic signal anchor proteins is specifically impaired, while highly hydrophobic sequences can still be efficiently inserted through the SRP pathway .

What are effective methods for studying SEC62 function in C. albicans?

To study SEC62 function in C. albicans, researchers can generate conditional mutant strains by placing SEC62 under the control of a tetracycline-regulated promoter (similar to approaches used for SEC6) . This allows for controlled repression of SEC62 expression and observation of resulting phenotypes before secondary effects develop. When analyzing SEC62 function, it's important to examine multiple cellular processes including cytokinesis, endocytosis, secretion of virulence factors (like aspartyl proteases and lipases), and hyphal morphogenesis. Electron microscopy can be used to visualize accumulation of secretory vesicles and endosomal structures . Additional approaches include co-immunoprecipitation to study protein-protein interactions and complementation studies with specific SEC62 domains to map functional regions.

How can researchers analyze the interaction between SEC62 and SEC63?

The interaction between SEC62 and SEC63 can be studied using co-immunoprecipitation assays with epitope-tagged proteins. For example, FLAG-tagged SEC62 variants can be expressed in cells containing HA-tagged SEC63, followed by isolation of crude membranes and immunoprecipitation with anti-FLAG antibodies . This approach can reveal how mutations in SEC62, particularly in the N-terminal domain containing positively charged residues, affect its interaction with SEC63. Studies have shown that substituting positively charged residues with negatively charged or even neutral residues (such as at positions 35-37 or 103-105) disrupts the electrostatic interaction between the N-terminus of SEC62 and the C-terminus of SEC63, impairing the formation of the SEC62-SEC63 complex .

What approaches can be used to investigate SEC62's role in membrane protein topogenesis?

To investigate SEC62's role in membrane protein topogenesis, researchers can use engineered model proteins with signal anchor sequences of varying hydrophobicity. For example, researchers have created H1 signal anchor proteins (such as H1-4L and H1-5L) and expressed them in wild-type and SEC62 mutant strains to assess how SEC62 affects membrane insertion and orientation . By analyzing the translocation of the C-terminus of these membrane proteins in various SEC62 mutants (such as those with alterations in the N-terminal domain), researchers can determine how SEC62 contributes to establishing proper membrane protein topology. Protease protection assays, glycosylation analysis, and reporter enzyme activities can be used to assess translocation efficiency and protein orientation .

What key domains and residues in SEC62 are critical for its function?

Several key domains and residues in SEC62 are critical for its function:

• N-terminal cytosolic domain: Contains clusters of positively charged residues (positions 35-37 and 103-105) that are essential for the interaction with SEC63 and proper function .

• Transmembrane domains: Particularly TM2, which helps stabilize signal sequences at the lateral gate. Bulky hydrophobic residues in TM1 (positions 157-159) and TM2 (positions 185-187) contribute to membrane anchoring and possibly substrate interaction .

• C-terminal cytosolic domain: Following TM2, this domain is essential for cell viability. Residues 218-220 (FPN) are particularly important, as mutations in this region can be lethal .

Studies have shown that mutations replacing positively charged residues in the N-terminal domain with negatively charged or even neutral amino acids severely impair SEC62 function, particularly its ability to facilitate the translocation of moderately hydrophobic signal sequences .

How do mutations in different domains of SEC62 affect its functionality?

Mutations in different domains of SEC62 have distinct effects on its functionality:

Notably, double mutations combining N-terminal alterations (35DDD+103DDD) do not show additive defects, suggesting these domains may function in the same pathway or interaction .

What can we learn from SEC62 complementation studies in yeast?

Complementation studies in yeast provide valuable insights into SEC62 function. When various SEC62 mutants are transformed into a SEC62 deletion strain (using plasmid shuffling techniques), the ability of these variants to support cell viability reveals which domains and residues are essential . For example, all SEC62 variants except those with mutations in positions 218-220 (FPN→AAA) can functionally replace wild-type SEC62, indicating this C-terminal region is essential for viability. While mutations in the N-terminal domain (positions 35-37 or 103-105) support viability, they show functional defects in protein translocation, particularly for moderately hydrophobic signal sequences . These complementation studies also reveal that the role of SEC62 in membrane protein topogenesis is most significantly affected by mutations in the N-terminal domain that disrupt the SEC62-SEC63 interaction, highlighting the importance of this protein complex for proper membrane protein orientation.

What is the relationship between SEC62 and fungal cell wall formation?

SEC62, as part of the protein translocation machinery, plays an important role in fungal cell wall formation. The cell wall is a dynamic structure that requires continuous delivery of newly synthesized proteins and cell wall components through the secretory pathway. Studies on the related SEC6 protein in C. albicans have shown that defects in this pathway lead to aberrant localization of chitin at the septum and increased resistance to zymolyase activity, suggesting altered cell wall composition and architecture . As SEC62 mediates the translocation of proteins across the ER membrane, dysfunction of SEC62 would disrupt the trafficking and delivery of enzymes involved in cell wall synthesis and remodeling, as well as structural components of the cell wall. This would potentially alter cell wall integrity, composition, and the exposure of pathogen-associated molecular patterns (PAMPs) that interact with host immune cells.

Could SEC62 serve as a potential target for antifungal development?

SEC62 represents a potential target for antifungal development for several reasons:

• It is essential for cell viability, as demonstrated by complementation studies

• It has specific domains and interactions, particularly the SEC62-SEC63 complex, that could be targeted by small molecule inhibitors

• It contributes to multiple virulence-associated processes including secretion of virulence factors, cell wall formation, and potentially hyphal morphogenesis

• While conserved among fungi, there may be structural or functional differences from mammalian homologs that could allow for selective targeting

How does SEC62 function differ in post-translational versus co-translational translocation?

SEC62 functions primarily in the post-translational translocation pathway, where fully synthesized proteins are transported across the ER membrane after completion of protein synthesis . In this context, SEC62 works alongside SEC63, SEC71, and SEC72 to recognize and translocate specific substrates, particularly those with moderately hydrophobic signal sequences . The post-translational mode contrasts with the co-translational pathway, which relies on the Signal Recognition Particle (SRP) and typically handles proteins with more hydrophobic signal sequences .

Interestingly, research suggests there is some overlap between these pathways. Defects in SRP can impair translocation of signal anchor proteins across the hydrophobicity spectrum, while defects in SEC62 specifically impair moderately hydrophobic signal sequences . This indicates that moderately hydrophobic signal sequences may require both pathways for optimal targeting and translocation. SEC62's role seems particularly important for stabilizing less hydrophobic signal sequences at the lateral gate of the SEC61 translocon, a function that may be less critical for highly hydrophobic sequences that can be efficiently recognized and stabilized by the SRP-dependent pathway .

What are the critical controls needed when investigating SEC62 function using conditional mutant strains?

When investigating SEC62 function using conditional mutant strains, several critical controls are necessary:

• Expression verification: Monitor SEC62 expression levels (both mRNA and protein) throughout the experiment to confirm appropriate repression

• Viability assessment: Since SEC62 is essential, ensure observed phenotypes aren't due to general cellular dysfunction by monitoring cell viability (conditional SEC62 mutants may remain viable for only a limited time, e.g., up to 27 hours as observed with SEC6)

• Timing controls: Perform all phenotypic analyses before viability is compromised (e.g., at 24 hours or earlier if using a similar system to the tetR-SEC6 mutant)

• Wild-type comparisons: Include parallel experiments with wild-type strains subjected to the same conditions

• Phenotypic specificity: Examine multiple cellular processes (secretion, endocytosis, cell wall formation, hyphal morphogenesis) to distinguish primary from secondary effects

• Rescue experiments: Complement the conditional mutant with wild-type SEC62 to confirm phenotypes are specifically due to SEC62 dysfunction

• Microscopic verification: Use electron microscopy to confirm the accumulation of specific vesicle populations, which can help distinguish defects in different steps of the secretory pathway

How can researchers analyze the interaction between SEC62 and different signal sequences?

Researchers can analyze the interaction between SEC62 and different signal sequences through several approaches:

• Model protein analysis: Create a set of reporter proteins with signal anchor sequences of varying hydrophobicity (like the H1-4L and H1-5L constructs) and express them in wild-type and SEC62 mutant strains

• Mutational analysis: Introduce mutations in both SEC62 (particularly in the transmembrane domains) and signal sequences to map interaction determinants

• Cross-linking studies: Use chemical cross-linking followed by immunoprecipitation or mass spectrometry to identify direct contacts between SEC62 and substrate signal sequences

• In vitro reconstitution: Reconstitute the translocation system with purified components to directly assess SEC62-signal sequence interactions under controlled conditions

• Comparative analysis: Test signal sequences from different classes of proteins (secreted proteins, membrane proteins with different topologies) to determine if SEC62 has preferences beyond hydrophobicity

• Competitive assays: Determine whether different signal sequences compete for SEC62 binding, which would suggest a direct interaction

Through these approaches, researchers can determine the specificity and mechanisms by which SEC62 recognizes and facilitates the translocation of proteins with different signal sequences, particularly those with moderate hydrophobicity that appear to depend more heavily on SEC62 function .