Recombinant Saccharomyces cerevisiae Monopolar spindle protein 2 (MPS2)

What is the molecular structure and localization of the MPS2 protein?

MPS2 encodes an essential 44-kDa protein with two putative coiled-coil regions and a hydrophobic sequence. Biochemical fractionation experiments have conclusively demonstrated that MPS2 is an integral membrane protein. When visualized using either GFP fusion proteins in living cells or through indirect immunofluorescence microscopy of epitope-tagged versions (such as 9xmyc-MPS2), the protein exhibits a perinuclear localization pattern with one or two brighter foci of staining corresponding to the spindle pole body. Immunoelectron microscopy has further confirmed that GFP-MPS2 localizes specifically to the SPB .

Methodology for localization studies typically involves:

• Tagging MPS2 with fluorescent proteins (GFP) or epitope tags (myc)

• Visualization through fluorescence microscopy or immunofluorescence

• Confirmation with immunoelectron microscopy for precise subcellular localization

How essential is MPS2 for cellular viability in S. cerevisiae?

This survival mechanism appears to involve ploidy changes that may compensate for the loss of MPS2 function, suggesting complex genetic interactions that can partially bypass the requirement for this protein. This phenomenon has been observed with multiple independently constructed null alleles of MPS2 .

What are the most effective strategies for generating recombinant MPS2 constructs?

Based on published research, successful MPS2 cloning and expression strategies include:

• Genomic isolation: The MPS2 gene can be isolated from yeast genomic libraries (such as those constructed in centromeric plasmids like YCp50) by complementation screening of temperature-sensitive mutants (e.g., mps2-1) .

• Epitope tagging protocols:

  • N-terminal tagging: 9xmycN-MPS2 under control of its endogenous promoter

  • C-terminal tagging: 9xmycC-MPS2

  • GFP fusion proteins: GFP-MPS2 for visualization in living cells

• Expression systems:

  • Endogenous promoter expression for physiological studies

  • GAL1-regulated expression for overexpression experiments

  • CLB2 promoter replacement (PCLB2-MPS2) for meiosis-specific depletion

It's important to note that C-terminal tagging (9xmycC-MPS2) may cause mild functional defects, as cells expressing this construct can spontaneously increase in ploidy, suggesting careful validation of tagged constructs is essential .

How can researchers effectively study MPS2 function through mutant analysis?

Research on MPS2 function has employed several mutant-based strategies:

• Temperature-sensitive mutants: The mps2-1 allele contains a single base change at nucleotide 114, resulting in a glutamic acid to lysine substitution at position 39. This mutant exhibits SPB duplication defects at restrictive temperature (37°C), with the nascent SPB failing to insert into the nuclear envelope .

• Null allele construction: Complete gene replacement with marker genes (HIS3) through homologous recombination.

• Meiosis-specific depletion: Using the CLB2 promoter replacement strategy (PCLB2-MPS2) to halt MPS2 production at the onset of meiosis.

• Domain analysis: Construction of deletion mutations targeting specific regions (coiled-coil domains, hydrophobic sequences) to identify functional domains.

How does MPS2 contribute to SPB duplication and function?

MPS2 is specifically required for a late step in SPB duplication, particularly for the insertion of the nascent SPB into the nuclear envelope. In temperature-sensitive mps2-1 mutants at restrictive temperature, cells contain duplicated SPBs, but the nascent SPB fails to be inserted into the nuclear envelope. Instead, the defective SPB remains on the cytoplasmic face of the NE, unable to nucleate nuclear microtubules .

The molecular mechanism involves:

• MPS2 acts as an integral membrane component of the SPB

• Its membrane-spanning domains likely facilitate the integration of the nascent SPB into the nuclear envelope

• Failure of this insertion process leads to monopolar spindles and chromosome segregation defects

• This results in G2 cell cycle arrest with large buds and unsegregated DNA

Research indicates that MPS2 functions in conjunction with NDC1, as ndc1-1 mutant strains exhibit phenotypes indistinguishable from mps2-1 strains at non-permissive temperature .

Is MPS2 expression or function regulated during the cell cycle?

Experimental evidence suggests that MPS2 protein levels remain relatively constant throughout the cell cycle. Studies tracking 9xmycN-MPS2 under the control of its endogenous promoter in synchronized cells (released from α-factor arrest) show that levels of MPS2 do not significantly oscillate compared to the invariant control protein Cdc28p. There may be a slight decrease during the G1-S phases, but this is substantially less dramatic than the complete disappearance observed with cell-cycle regulated proteins like Pds1-HAp .

This relatively constant expression pattern suggests that:

• MPS2 function may be regulated post-translationally rather than through protein abundance changes

• The protein likely has a structural role at the SPB throughout the cell cycle

• Its activity might be modulated through interactions with other cell cycle-regulated proteins

What is known about MPS2's connection to the ubiquitin-proteasome pathway?

MPS2 was identified in a genetic screen for genes whose overexpression is toxic in a cim5 proteasome mutant at semi-permissive temperature but not in wild-type strains. This connection to the ubiquitin-proteasome pathway suggests potential regulation mechanisms that remain to be fully elucidated .

Experimental approaches to investigate this connection include:

• Genetic interaction studies: Testing synthetic interactions between MPS2 and components of the ubiquitin-proteasome system

• Protein stability analysis: Measuring MPS2 protein half-life in proteasome mutants versus wild-type cells

• Ubiquitination assays: Detecting possible ubiquitinated forms of MPS2 through immunoprecipitation and Western blotting

Research questions remain about whether MPS2 itself is a proteasome substrate or whether it functions in a pathway that becomes essential when proteasome function is compromised.

How do the coiled-coil domains of MPS2 contribute to its function and protein interactions?

MPS2 contains two putative coiled-coil regions that likely mediate protein-protein interactions essential for its function. While the specific interaction partners for these domains remain to be fully characterized, experimental approaches to study their function include:

The presence of these structural motifs suggests MPS2 may function as part of a larger protein complex at the SPB, potentially forming oligomers or interacting with other SPB components through these domains.

What methodologies can be used to investigate MPS2's role in meiosis?

MPS2 has been implicated in meiotic progression, with meiotic depletion resulting in defects in meiosis I. When the endogenous MPS2 promoter is replaced by that of CLB2 (PCLB2-MPS2), production of MPS2 is halted at the onset of meiosis. While more than 60% of these cells can initiate meiosis I (as determined by Tub4-mApple foci separation), less than 5% complete meiosis to form four Tub4-mApple foci, compared to more than 80% of wild-type cells .

Recommended methodologies for investigating MPS2's meiotic function include:

• Meiosis-specific depletion: Using promoter replacement strategies like PCLB2-MPS2

• Live cell imaging: Tracking fluorescently tagged markers (Tub4-mApple) through meiotic progression

• Chromosome segregation analysis: Monitoring DNA segregation patterns using DAPI staining or fluorescent chromosome markers

• Synchronization techniques: Inducing synchronized meiosis to allow precise temporal analysis

• Telomere dynamics visualization: Investigating potential roles in telomere-associated LINC (Linker of Nucleoskeleton and Cytoskeleton) complex formation during meiosis

What are the most common pitfalls when working with MPS2 and how can they be avoided?

Research with MPS2 presents several technical challenges:

• Genetic manipulation complications:

  • Lethality of null mutations requires maintaining the gene on a plasmid during strain construction

  • Solution: Use plasmid shuffle techniques with 5-FOA counterselection or temperature-sensitive alleles

• Tagging interference:

  • C-terminal tagging (9xmycC-MPS2) can cause mild functional defects

  • Solution: Use N-terminal tagging or internal tagging approaches, always validating functionality

• Spontaneous suppressors:

  • MPS2 null or mutant strains may acquire suppressor mutations that mask phenotypes

  • Solution: Use freshly constructed strains and monitor for phenotypic changes over time

• Ploidy changes:

  • MPS2 mutants frequently undergo ploidy changes that can confound experiments

  • Solution: Regularly check ploidy by flow cytometry or microscopic measurement of cell/nuclear size

How can researchers differentiate between direct and indirect effects of MPS2 mutations?

Distinguishing primary from secondary effects of MPS2 mutations requires careful experimental design:

• Rapid inactivation approaches:

  • Use of temperature-sensitive alleles with quick temperature shifts

  • Auxin-inducible degron (AID) tagging for rapid protein depletion

  • Anchor-away techniques to rapidly relocalize the protein

• Time-course analysis:

  • Tracking multiple cellular processes with temporal resolution after MPS2 inactivation

  • Establishing the sequence of events to determine primary defects

• Separating phenotypes:

  • Construction of allelic series with distinct phenotypes

  • Targeted mutations affecting specific protein domains or functions

• Suppressor analysis:

  • Identification of genetic suppressors can reveal pathway relationships

  • Distinguishing bypass suppressors from direct interaction partners

For example, the role of MPS2 in SPB insertion can be distinguished from its effects on cell cycle progression by combining temperature-sensitive mutations with cell cycle arrest at different stages.