Recombinant Saccharomyces cerevisiae Inner nuclear membrane protein HEH2 (HEH2)

What is HEH2 and where is it localized in yeast cells?

HEH2 (also known as Man1) is an integral membrane protein of the Lap2-emerin-MAN1 (LEM) family in Saccharomyces cerevisiae. It is specifically localized to the inner nuclear membrane (INM) and plays important roles in maintaining the functional and physical integrity of the nuclear envelope. The protein contains specific domains that enable both its targeting to the inner nuclear membrane and its interactions with other nuclear components, particularly nuclear pore complexes (NPCs) . HEH2 is part of a wider family of LEM domain proteins that have emerged as crucial components of nuclear envelope structure and function.

What are the key structural domains of HEH2 protein?

HEH2 protein contains several distinct functional domains:

• N-terminal domain: Required for targeting to the inner nuclear membrane (INM)

• LEM domain: Characteristic of the Lap2-emerin-MAN1 family

• Nuclear localization signal (NLS): Adopts an IBB-like fold necessary for import to the INM

• Intrinsically disordered linker region (L): Creates distance between the NLS and transmembrane segment, forming part of the NLS-L-TM domain composition essential for proper localization

• Transmembrane (TM) segment: Anchors the protein in the membrane

• C-terminal winged helix (WH) domain: Mediates stable interactions with the nuclear pore complex

This domain organization allows HEH2 to perform its specialized functions at the nuclear envelope while maintaining proper localization.

How is HEH2 evolutionarily related to other LEM domain proteins?

Despite their independent evolutionary origins (with approximately 500 million years of separation), both ScHeh2 and SpHeh2 have developed functional connections with nuclear pore complexes. This represents a fascinating case of convergent evolution where proteins with distinct evolutionary histories have acquired similar functions. The affinity purifications of SpHeh2-TAP and ScHeh2-TAP show qualitatively similar interaction profiles, particularly with components of the inner ring complex of nuclear pore complexes .

How does HEH2 interact with the nuclear pore complex (NPC)?

HEH2 demonstrates specific and evolutionarily conserved interactions with the nuclear pore complex, particularly with components of the inner ring complex (IRC). This interaction is primarily mediated by the C-terminal winged helix (WH) domain of HEH2, which is functionally distinct from the N-terminal domain required for INM targeting .

The interaction network identified through affinity purification studies reveals that HEH2 copurifies with several specific nuclear pore proteins. In S. cerevisiae, the Heh2 complex consists of a subset of inner ring nucleoporins including Nup192, Nup188, Nup170, Pom152, Nup59, Nup53, and Nup157 . Similarly, in S. pombe, despite the distinct evolutionary origin of SpHeh2, it associates with homologous nucleoporins including Nup184, Nup186, Nup155, Pom152, Npp106, Nup98, and Nup97 .

This interaction appears to be functionally significant, as inhibiting Heh2's interactions with the NPC by deleting the Heh2 WH domain leads to NPC clustering, suggesting a role in NPC distribution or assembly .

What is the mechanism of HEH2 targeting to the inner nuclear membrane?

The targeting of HEH2 to the inner nuclear membrane involves an active transport mechanism that depends on several key elements:

• Nuclear localization signal (NLS): HEH2 contains an NLS that adopts an IBB-like fold, which is essential for its import to the INM .

• Intrinsically disordered linker region (L): This creates necessary distance between the NLS and the transmembrane segment, forming the crucial NLS-L-TM domain composition .

• Recognition by nuclear transport machinery: The NLS is recognized by importin α/β, facilitating active transport through nuclear pore complexes.

Interestingly, this mechanism appears to be evolutionarily conserved. Studies have shown that the nuclear localization signal of metazoan Pom121 shares biochemical, structural, and functional properties with those of Heh1 and Heh2. Additionally, a Heh2-derived reporter protein has been demonstrated to successfully target to the inner membrane in human Hek293T cells . This suggests that the active import mechanism for INM proteins may be conserved across species but is likely confined to a small subset of inner nuclear membrane proteins.

How do mutations in the HEH2 WH domain affect nuclear pore complex distribution?

Mutations in the HEH2 winged helix (WH) domain have significant effects on nuclear pore complex organization. Specifically, deletion of the Heh2 WH domain leads to NPC clustering, indicating that the interaction between Heh2 and the NPC through this domain is critical for proper NPC distribution across the nuclear envelope .

Research suggests that there are two distinct pools of Heh2: one associated with NPCs and another distributed throughout the INM. Inhibition of NPC assembly, for example by trapping newly synthesized Nup192-FRB-GFP (a key NPC component), reduces the pool of Heh2 bound to NPCs . This supports a model where Heh2 may differentially interact with NPC assembly intermediates compared to fully formed NPCs, potentially playing a role in NPC biogenesis or maintenance.

The functional significance of this interaction is further supported by synthetic genetic interactions between genes encoding NPC components (nucleoporins) and HEH2, suggesting cooperative roles in cellular processes .

What are the recommended methods for studying HEH2 localization in yeast cells?

For studying HEH2 localization in yeast cells, researchers should consider the following methodological approaches:

When designing these experiments, it's crucial to incorporate proper controls and randomize experimental runs to protect against unknown nuisance variables . Additionally, using multiple complementary approaches strengthens the reliability of localization findings.

How can researchers effectively purify and characterize HEH2 protein complexes?

Effective purification and characterization of HEH2 protein complexes can be achieved through the following methods:

• Tandem Affinity Purification (TAP):

  • Tag HEH2 with a TAP tag (as demonstrated in studies with SpHeh2-TAP and ScHeh2-TAP)

  • Two-step purification process to achieve high purity

  • Note that SpHeh2-TAP is proteolytically sensitive and purifies as both full-length (~115 kDa) and smaller (~65 kDa) forms

• Mass spectrometry analysis:

  • Identify co-purifying proteins to determine interaction partners

  • Compare with control purifications to identify specific interactors

• Co-immunoprecipitation:

  • Use antibodies against HEH2 or its tag for immunoprecipitation

  • Western blotting to detect specific interaction partners

• Size exclusion chromatography:

  • Separate protein complexes based on size

  • Can be coupled with multi-angle light scattering for molecular weight determination

• Crosslinking approaches:

  • Chemical crosslinking followed by mass spectrometry to identify proteins in close proximity to HEH2

  • Helps define the architecture of protein complexes

When analyzing interaction data, researchers should consider the evolutionary conservation of interactions. For example, compare interaction profiles between S. cerevisiae and S. pombe HEH2 to identify conserved partners, which may represent functionally important interactions .

What experimental design approaches are most effective for studying HEH2 function?

For studying HEH2 function, researchers should implement rigorous experimental design principles:

• Define clear objectives: Determine specific response variables that can be measured to assess HEH2 function .

• Identify relevant factors:

  • Controllable factors: Gene mutations, expression levels, growth conditions

  • Uncontrollable variables: Account for known nuisance variables through blocking and randomize runs to protect against unknown variables

• Design genetic studies:

  • Gene deletion/mutation analysis: Systematic mutation of HEH2 domains

  • Synthetic genetic interaction screens: Identify genes that interact with HEH2 (as demonstrated by synthetic genetic interactions between NPC components and HEH2)

  • Suppressor screens: Identify mutations that rescue HEH2 mutant phenotypes

• Structure-function analysis:

  • Domain swap experiments: Exchange domains between HEH2 and related proteins

  • Cross-species complementation: Test if HEH2 from other species can functionally replace S. cerevisiae HEH2

• Functional assays:

  • Nuclear envelope integrity assays

  • NPC clustering quantification

  • Nuclear import/export assays

  • Cell cycle progression analysis

• Statistical considerations:

  • Determine appropriate sample sizes based on variability estimates and desired precision

  • Reserve resources for follow-up experiments (recommended: use only 25% of resources in the first experiment)

  • Perform pro forma analysis with response variables as random variables to check for estimability of factor effects and precision

By systematically applying these approaches, researchers can effectively dissect the various functions of HEH2 and its role in nuclear envelope organization.

How does HEH2 contribute to nuclear envelope integrity and function?

HEH2, as a member of the LEM (Lap2-emerin-MAN1) family of integral membrane proteins, plays crucial roles in maintaining nuclear envelope integrity and function through several mechanisms:

• Nuclear pore complex organization: HEH2 interacts with the inner ring complex of nuclear pore complexes, and disruption of this interaction (through deletion of the WH domain) leads to NPC clustering . This suggests HEH2 helps maintain proper NPC distribution across the nuclear envelope.

• NPC assembly or maintenance: There is evidence suggesting HEH2 may differentially bind to NPC assembly intermediates compared to fully formed NPCs . This indicates a potential role in NPC biogenesis or quality control.

• Structural support: As an inner nuclear membrane protein, HEH2 likely contributes to the physical integrity of the nuclear envelope through its membrane-spanning domains and interactions with other nuclear envelope components.

• Nuclear-cytoplasmic transport: Through its interactions with the NPC, HEH2 may influence nuclear transport processes, particularly for specific cargoes.

The functional importance of HEH2 is underscored by synthetic genetic interactions between genes encoding NPC components and HEH2 , indicating cooperative roles in essential cellular processes.

What is the relationship between HEH2 and chromosome organization?

While the provided search results don't directly address HEH2's role in chromosome organization, related research suggests potential functions:

• LEM domain proteins, including HEH2, typically interact with chromatin-associated proteins, potentially influencing chromosome organization.

• In the S. pombe cousin Schizosaccharomyces japonicus, it has been suggested that Heh2 supports connections between chromatin and NPCs to enable their segregation between daughter cells during mitosis . This indicates a potential role in chromosome dynamics during cell division.

• Nuclear envelope proteins often play roles in organizing the genome through anchoring of chromosomal regions at the nuclear periphery, which can influence gene expression and DNA repair processes.

Further research is needed to fully elucidate HEH2's specific contributions to chromosome organization in S. cerevisiae, including potential interactions with chromatin-modifying enzymes, structural chromosomal proteins, or specialized chromosome regions like telomeres or centromeres.

How conserved is the function of HEH2 across different yeast species?

Despite the distinct evolutionary origins of HEH2 in different yeast species, functional studies reveal remarkable conservation of its interactions and roles:

• Evolutionary divergence: S. cerevisiae and S. pombe are separated by approximately 500 million years of evolution . Interestingly, while their HEH1 genes (ScHEH1 and SpHEH1) are orthologues derived from a common ancestor, their HEH2 genes (ScHEH2 and SpHEH2) arose from independent duplication events of their respective HEH1 paralogues .

• Conserved interactions: Despite this unique evolutionary history, affinity purifications of SpHeh2-TAP and ScHeh2-TAP show qualitatively similar interaction profiles . Both proteins specifically copurify with homologous components of the nuclear pore complex inner ring.

• Similar biochemical properties: In both species, HEH2 copurifies with a specific subset of inner ring nucleoporins, suggesting conservation of binding interfaces and functional interactions .

• Functional conservation with higher eukaryotes: The active transport mechanism for inner nuclear membrane targeting appears conserved between yeast and metazoans. The nuclear localization signal of human Pom121 shares biochemical, structural, and functional properties with those of yeast Heh1 and Heh2 . Additionally, a Heh2-derived reporter protein can successfully target to the inner membrane in human HEK293T cells .

This functional conservation despite divergent evolutionary origins represents a fascinating example of convergent evolution, where similar molecular mechanisms have evolved independently to fulfill comparable cellular functions.

What are the most promising future research directions for HEH2 studies?

Several promising research directions could significantly advance our understanding of HEH2 function:

• Structural biology approaches: Determining the three-dimensional structure of HEH2 domains, particularly the winged helix (WH) domain and its interaction with nuclear pore components, would provide mechanistic insights into function.

• Dynamic studies: Investigating the temporal aspects of HEH2 function during the cell cycle, particularly during nuclear envelope breakdown and reformation in mitosis, would clarify its role in nuclear architecture maintenance.

• Genome-wide screens: Comprehensive genetic interaction screens to identify the full network of genes functionally connected to HEH2.

• Cross-species functional analysis: Further exploration of functional conservation between evolutionary distant HEH2 proteins, including testing cross-species complementation.

• Mechanistic studies of NPC assembly: Investigating whether HEH2 directly participates in NPC assembly, possibly as a scaffold or quality control factor.

• Chromatin interactions: Mapping potential interactions between HEH2 and chromatin, which could reveal roles in gene expression regulation.

• Development of HEH2 as a tool: Exploring the potential use of HEH2 domains for targeting proteins to the inner nuclear membrane in synthetic biology applications.