Mad1 is a non-essential

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

which in

yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are est ...

has a function in the spindle assembly checkpoint (SAC). This checkpoint monitors chromosome attachment to spindle microtubules and prevents cells from starting anaphase until the spindle is built up. The name Mad refers to the observation that mutant cells are mitotic arrest deficient (MAD) during microtubule depolymerization. Mad1 recruits the anaphase inhibitor Mad2 to unattached kinetochores and is essential for Mad2-

Cdc20 The cell division cycle protein 20 homolog is an essential regulator of cell division that is encoded by the ''CDC20'' gene in humans. To the best of current knowledge its most important function is to activate the anaphase promoting complex (AP ...

complex formation ''in vivo'' but not ''in vitro''. ''In vivo'', Mad1 acts as a competitive inhibitor of the Mad2-Cdc20 complex. Mad1 is phosphorylated by Mps1 which then leads together with other activities to the formation of the mitotic checkpoint complex (MCC). Thereby it inhibits the activity of the anaphase-promoting complex/cyclosome (APC/C). Homologues of Mad1 are conserved in eukaryotes from yeast to mammals.

Introduction

In the early 90s, yeast genes were identified which mutations resulted in a defect in mitotic arrest in response to microtubule disassembly (mitotic arrest deficient genes - MAD genes). These cells showed no mitotic arrest in the presence of microtubule polymerization inhibitors and were therefore not able to delay cell division. The genes identified included the ''MAD1'', '' MAD2'' and '' MAD3'' genes. They are conserved in all

eukaryotes The eukaryotes ( ) constitute the domain of Eukaryota or Eukarya, organisms whose cells have a membrane-bound nucleus. All animals, plants, fungi, seaweeds, and many unicellular organisms are eukaryotes. They constitute a major group of ...

and are involved in a pathway that is active in

prometaphase Prometaphase is the phase of mitosis following prophase and preceding metaphase in eukaryotic somatic cells. In prometaphase, the nuclear membrane breaks apart into numerous "membrane vesicles," and the chromosomes inside form protein structure ...

to prevent the premature separation of sister

chromatids A chromatid (Greek ''khrōmat-'' 'color' + ''-id'') is one half of a duplicated chromosome. Before replication, one chromosome is composed of one DNA molecule. In replication, the DNA molecule is copied, and the two molecules are known as chrom ...

and constitute the so-called spindle assembly checkpoint (SAC). This checkpoint monitors the status of chromosome attachment to the mitotic spindle and inhibits the

metaphase Metaphase ( and ) is a stage of mitosis in the eukaryotic cell cycle in which chromosomes are at their second-most condensed and coiled stage (they are at their most condensed in anaphase). These chromosomes, carrying genetic information, alig ...

anaphase Anaphase () is the stage of mitosis after the process of metaphase, when replicated chromosomes are split and the newly-copied chromosomes (daughter chromatids) are moved to opposite poles of the cell. Chromosomes also reach their overall maxim ...

transition by preventing the activation of the

anaphase-promoting complex Anaphase-promoting complex (also called the cyclosome or APC/C) is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The APC/C is a large complex of 11–13 subunit proteins, including a cullin ...

/cyclosome (APC/C), and thereby the degradation of

cell cycle The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell (biology), cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA (DNA re ...

regulators. Mad1 in this pathway accumulates at unattached kinetochores and acts as a sensor for unattached kinetochores in this machinery.

Function

Eukaryotic cells show a mitotic arrest in the presence of microtubule polymerization inhibitors. A spindle assembly checkpoint monitors the status of the spindle and links the metaphase-anaphase transition to proper bipolar attachment of all kinetochores to the mitotic spindle. The spindle assembly checkpoint inhibits the activity of the anaphase promoting complex by preventing degradation of downstream effectors, which otherwise lead to anaphase onset and exit from mitosis. Depletion of Mad1 leads to the loss of SAC function. Mad1 localises predominantly at unattached kinetochores and triggers mitotic arrest in case of a single unattached kinetochore. Mad1 recruits the important SAC component Mad2 to unattached kinetochores and induces mitotic arrest signal amplification. There is a pool of free cytoplasmic Mad2 in its inactive open conformation called o-MAD2. When bound to Mad1, Mad2 adopts an active conformation called closed (c-Mad2) and forms a heterotetramer of two Mad1 and two c-Mad2 units. The heterotetramer of Mad1–c-Mad2 is very stable and works as a

catalytic Catalysis () is the increase in reaction rate, rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst ...

receptor Receptor may refer to: * Sensory receptor, in physiology, any neurite structure that, on receiving environmental stimuli, produces an informative nerve impulse *Receptor (biochemistry), in biochemistry, a protein molecule that receives and respond ...

for free cytoplasmic o-Mad2. Free o-Mad2 binds to this receptor and changes its conformation to the active closed form. This second c-MAD2 is transferred to

with yet unknown mechanism and forms Cdc20–c-Mad2 complex. This complex is an essential component of mitotic checkpoint complex (MCC). MCC binds and inhibits APC/C and therefore arrests progression through mitosis.

Regulation

There are two upstream checkpoint

kinases In biochemistry, a kinase () is an enzyme that catalysis, catalyzes the transfer of phosphate groups from High-energy phosphate, high-energy, phosphate-donating molecules to specific Substrate (biochemistry), substrates. This process is known as ...

implicated in regulating Mad1 function through

phosphorylation In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...

. Mps1 phosphorylates Mad1 both ''in vitro'' and ''in vivo'' and is thought to regulate Mad1 and Mad2 localization to

kinetochores A kinetochore (, ) is a flared oblique-shaped protein structure associated with duplicated chromatids in eukaryotic cells where the spindle fibers, which can be thought of as the ropes pulling chromosomes apart, attach during cell division to p ...

and their interaction dynamics.

BUB1 Mitotic checkpoint serine/threonine-protein kinase BUB1 also known as BUB1 (budding uninhibited by benzimidazoles 1) is an enzyme that in humans is encoded by the ''BUB1'' gene. Bub1 is a serine/threonine protein kinase first identified in genet ...

is the other kinase that recruits Mad1 to kinetochores and activates it if a kinetochore is unattached. If a kinetochore is attached to spindle, SAC inhibitor p31^comet inhibits Mad1 mediated conformational rearrangement of Mad2 and prevents Mad2 from binding to Cdc20.

Structural features and mechanism

By biochemical methods Mad1 was predicted to encode a 90kD, 718-residue, coiled-coil

with a characteristic rod shape in 1995. Crystal structures followed soon. Then in 2002 the crystal structure of human Mad1 in complex with human Mad2 forming a tetramer was published. Due to experimental limitations the structure only shows Mad1 residues 484 - 584. Elongated Mad1 monomers are tightly held together by a parallel coiled-coil involving the N-terminal alpha helices. The Mad1 chains point away from the coiled-coil towards their Mad2 ligands forming two sub-complexes with Mad2. The segment between alpha helices 1 and 2 contains the Mad2 binding domain. The first part of this binding domain is flexible and adopts different conformations giving rise to an asymmetric complex. In their work, employing thermodynamic studies, Sironi et al. show that Mad1 functions such as to slow down the rate of Mad2-

complex formation and therefore acts as a competitive inhibitor ''in vivo''. Furthermore the authors suggest, the Mad1-Mad2 binding sites are buried inside the structure perhaps rendering the binding sites inaccessible for Cdc20 binding. Mad1-Mad2 binding is unusual in that the Mad2 C-terminal folds over Mad1. The authors therefore conclude that an unperturbed Mad1-Mad2 complex will not release Mad2 requiring a novel, so far poorly understood, mechanism of conformational change.

Cancer

Mismatches in chromosome number (aneuploidies) during meiosis are responsible for human diseases like Down's syndrome and also emerge frequently in cancer cells. The essential function of SAC gives rise to the hypothesis that mutations of the SAC and especially inactivation of SAC might be a reason for tumorigenesis or at least facilitate tumorigenesis. Against this idea, it was shown that cancer cells undergo apoptosis when components of the SAC are not present. In this model, in contrast to the other model, SAC inactivation becomes a potential way to kill rapidly dividing cancer cells. The molecular links between Mad1p, the SAC, apoptosis and cancer are still not fully understood.

References