LEY NÚMERO 12.318 DE ALIENACIÓN PARENTAL

In the search of the function of Pop3,1 decided to investigate w hether Pop3 has any involvem ent in proteolysis like P opl. Previous studies have established three main reasons for polyploidisation, which are linked to proteolysis.

1 / R u m l accum ulation - R um l (Moreno and N urse, 1994)is a CDK inhibitor, whose presence is required through G1 - phase and its destruction is needed to enter M-phase. If R um l is present at the end of S-phase, M -phase entry does not occur. H ence R u m l levels are closely regulated th ro u g h o u t the cell cycle, proteolysis being the major m echanism of regulation (Correa-Bordes and Nurse, 1995; M artin-Castellanos et a l, 1996). R um l is ubiquitin tagged by the SCF (Popl and Pop2) (Kominami et a l, 1998a; Kominami and Toda, 1997) and subsequently recognised and destroyed by proteosomes. Therefore, if R um l is not destroyed, the cell does not enter M -phase, instead it undergoes several round of S-phase, producing polyploid cells. Apopl and Apop2 m utants w ere show n to accum ulate non-ubiquinated R um l that was not recognised and destroyed by the proteasom e (Kominami et ah, 1998a; Kominami and Toda, 1997).

2 /C d c l8 accum ulation- C dcl8 is an activator of DNA replication, w hich is required through S - phase. Two m echanism s, transcription and proteolysis, regulate C dcl8 levels. C dcl8 is transcribed just before S-phase and it is degraded before entry of the cell into M - phase. C dcl8 is also a target of the SCF (Popl and Pop2) complex during or after S- phase, w hich ubiquinates it for degradation. Bypass of M - phase and several round of S - phase are produced w hen C dcl8 accum ulates, producing polyploid cells. Like R um l, A popl and Apop2 m utants accum ulate non- ubiquitinated C dcl8 (Jallepalli and Kelly, 1996; Kominami et al., 1998a; Kominami and Toda, 1997; Wolf et a l, 1999).

3/T h e third w ay in which polyploidisation m ay occur comprises several different phenotypes. The previous two reasons w ere due to cell cycle related proteins.

Pop3 & Cell Cycle regulation H ow ever, this third category, which I call C ut phenotype, contains m utants that are unable to segregate chromatids or that are unable to perform cytokinesis.

After replication of DNA the rod shape cells elongate, cells continue to grow and enter m itosis w hen they reach a certain size (Fantes, 1977a; Fantes, 1977b; N urse, 1975). D uring mitosis, sister chrom atids are pulled to opposite poles. A septum (cell wall) forms in the m iddle of the cell to separate it in two daughter cells

Two m ain phenotypes are seen in this group. M utants that show a disrupted and elongated nuclear phenotype due to the inability to segregate sister chrom atids; ciitl (Creanor and Mitchison, 1990; Funabiki et al, 1996a; Uzawa et al., 1990) and cut! (Hirano, 1986; (Funabiki et ah, 1996a; Funabiki et ah, 1996b; Uzawa et ah, 1990), these m utants show anaphase defects as the sister chrom atids can not separate and the chrom atin is "cut" off by the septum . The other category of the m utants undergo DNA replication and chrom osom e segregation b u t can not carry out cytokinesis, displaying elongated cells that contain m ultiple nuclei w ithout a septa that should divide the cell in two {cdc7) (Gould and Simanis, 1997). These cells show a 4C DNA content in the flow cytom etry analysis b u t they are not true polyploids, as the DNA content is due to several nuclei.

These three reasons are all connected w ith proteolysis. H ow ever the link betw een them is not to the same proteolytic machinery. In the study of Apopl (Jallepalli and Kelly, 1996; Kominami et ah, 1998a; Kominami and Toda, 1997; Wolf et ah, 1999), diploïdisation w as show n to be due to the accum ulation of R u m l and C dcl8 proteins. The accumulation in Apopl was due to a partial lack of ubiquitination in the cell, due to P o p l being a member of the SCF complex. The lack of P o p l results in accum ulation of large am ounts of n o n - ubiquitinated R um l and to a lesser extent C dcl8 proteins that are not recognised by the proteolytic m achinery and hence left undegraded. Pop2, another F-box protein in S. pombe (with hom ology to P o p l) has also been show n to function in the SCF complex. Apopl produces polyploid cells, w hich accum ulate R um l and C dcl8; Pop2 seem s to be m ore

Pop3 & Cell Cycle regulation specific for CdclS (Jallepalli and Kelly, 1996; Kominami et al, 1998a; W olf at ah, 1999).

C u tl and Cut2 (Funabiki at al., 1996a; Funabiki at aL, 1996b) studies, on the other h an d , have show n the need of the proteolytic cyclosom e/A P C (anaphase prom oting complex) (Sudankin at a l, 1995) complex for Cut2 deg rad atio n and hence, for the sister chromatid segregation.

Through the m orphological and m olecular characterisation several sim ilarities between Apopl and Apop3 m utants were found, which led us to investigate w hether Apop3 becomes polyploidised for similar reasons to Apopl or Apop2.