Skp1 and the F-box Protein Pof6 Are Essential for Cell Separation in Fission Yeast

Abstract

Here we report functional characterization of the essential fission yeast Skp1 homologue. We have created a conditional allele of skp1 (skp1-3f) mimicking the mutation in the budding yeast skp1-3 allele. Although budding yeast skp1-3 arrests at the G 1 /S transition, skp1-3f cells progress through S phase and instead display two distinct phenotypes. A fraction of the skp1-3f cells arrest in mitosis with high Cdc2 activity. Other skp1-3f cells as well as the skp1-deleted cells accumulate abnormal thick septa leading to defects in cell separation. Subsequent identification of 16 fission yeast F-box proteins led to identification of the product of pof6 (for pombe F-box) as a Skp1-associated protein. Interestingly, cells deleted for the essential pof6 gene display a similar cell separation defect noted in skp1 mutants, and Pof6 localizes to septa and cell tips. Purification of Pof6 demonstrates association of Skp1, whereas the Pcu1 cullin was absent from the complex. These findings reveal an essential non-Skp1-Cdc53/Cullin-F-box protein function for the fission yeast Skp1 homologue and the F-box protein Pof6 in cell separation.

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

“…Measurement of b-galactosidase activity demonstrated an interaction of similar strength between Mcs2 and Skp1 when compared to the interaction between Mcs2 and Mcs6. Most importantly, the interaction between Mcs2 and Skp1 was almost completely abolished when a Skp1 mutant (skp1-3) [40] with a single amino acid change (I172R) was used (Fig. 1B).…”

“…We recently reported on the identification and characterization of the fission yeast Skp1 [40], an essential subunit of the Skp1-Cullin-F box protein (SCF) ubiquitin ligase [40,41]. The original identification of Skp1 was made in a yeast two-hybrid screen using the Mcs2 (LexA-Mcs2) cyclin as bait (Fig.…”

“…We followed the levels of all three CAK subunits in Skp1 (skp1-3 [40]) or proteasome (mts3-1 [51]) temperature sensitive mutants, where known targets (Cdc18 and Cig2) of SCF-mediated ubiquitinylation and proteolysis accumulate at the restrictive temperature [52][53][54][55]. Total levels of HA-Mcs2, Pmh1-TAP, and Mcs6-TAP were followed by Western blot (anti-Ha or PAP; see Materials and methods) in wild type, skp1-3 or mts3-1 mutant strains after shift to restrictive temperature (Figs.…”

“…This is interesting as Skp1 functions not only as part of SCF but also plays a role as a regulator of a variety of complexes and functions including cell separation [40], recycling of proteins from the plasma membrane [59], and the assembly of vacuolar ATPases [47,60] apparently independently of SCF. Furthermore, our data suggest a potential new regulatory mechanism of Mcs2 and Pmh1 that could selectively regulate the distribution of these CAK subunits amongst the various cellular subcomplexes such as the trimeric versus TFIIH-associated forms of Mcs6.…”

Stepping into the ring: the SIN takes on contractile ring assembly: Figure 1.

“…S. pombe offers the opportunity to dissect in detail the entire cytokinetic pathway downstream from Plo1. In addition to clarifying the regulation of the SIN and Mid1 by Plo1, it will be important to identify the regulator(s) of formin Cdc12, as well as other factors important in formation and remodeling of the contractile ring, which may include ubiquitin ligases like the SCF/Pof6 complex (Hermand et al 2003), as well as actin cross-linking and depolymerizing factors (Nakano et al 2001;Wu et al 2001;Ono et al 2003;Nakano and Mabuchi 2006;Mukhina et al 2007). The regulation of these factors and their place in contractile ring formation, maintenance, and constriction will be of great interest in future studies.…”