Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast

Abstract

In interphase cells of fission yeast, the spindle pole body (SPB) is thought to be connected with chromosomal centromeres by an as yet unknown mechanism that spans the nuclear membrane. To elucidate this mechanism, we performed two-hybrid screens for proteins that interact with Kms1 and Sad1, which are constitutive membrane-bound components of the SPB that interact with each other. Seven and 26 genes were identified whose products potentially interact with Kms1 and Sad1, respectively. With the exception of Dlc1 (a homolog of the 14-kDa dynein light chain), all of the Kms1 interactors also interacted with Sad1. Among the genes identified were the previously known genes rhp9+ / crb2+, cut6+, ags1+ / mok1+, gst3+, kms2+, and sid4+. The products of kms2+ and sid4+ localize to the SPB. The novel genes were characterized by constructing disruption mutations and by localization of the gene products. Two of them, putative homologues of budding yeast UFE1 (which encodes a t-SNARE) and SFH1 (an essential component of a chromatin-remodeling complex), were essential for viability. Two further genes, which were only conditionally essential, genetically interact with sad1+. One of these was named sif1+ (for Sad1-interacting factor) and is required for proper septum formation at high temperature. Cells in which this gene was overexpressed displayed a wee -like phenotype. The product of the other gene, apm1+, is very similar to the medium chain of an adaptor protein complex in clathrin-coated vesicles. Apm1 appears to be required for SPB separation and spindle formation, and tended to accumulate at the SPB when it was overproduced. It was functionally distinct from its homologues Apm2 and Apm4. Other novel genes identified in this study included one for a nucleoporin and genes encoding novel membrane-bound proteins that were genetically related to Sad1. We found that none of the newly identified genes tested were necessary for centromere/telomere clustering.

Ppc89 Links Multiple Proteins, Including the Septation Initiation Network, to the Core of the Fission Yeast Spindle-Pole Body

“…Preliminary mass spectrometric analysis of a Ppc89 purification indicates that several coil-coil proteins including Pcp1 (Flory et al, 2002), Kms2 (Miki et al, 2004), and Cut12 (Bridge et al, 1998) copurify (unpublished observation). These are strong candidates for additional direct binding partners and studies are underway to test this possibility.…”

“…Sad1 contains one transmembrane helix domain and is required for mitotic functions of the SPB (Hagan and Yanagida, 1995). Interacting with Sad1 are Kms1 and Kms2, which contain coil-coiled and transmembrane helices and are not essential for vegetative growth (Niwa et al, 2000;Miki et al, 2004). Kms1 is essential for telomere clustering and SPB function during meiosis (Shimanuki et al, 1997).…”

“…Kms1 is essential for telomere clustering and SPB function during meiosis (Shimanuki et al, 1997). The less well-characterized kms2 mutant is reported to have mitotic defects (Miki et al, 2004). Another group of SPB proteins has been identified based on their specific requirement during meiosis.…”

Nuclear envelope attachment is not necessary for telomere function in fission yeast

“…1B). 4,12,13 In the kms1 can provide Sad1 with additional attachment sites on the nuclear envelope where telomeres locate. Indeed, multiple Sad1 foci can be seen on the nuclear envelope in addition to the SPB during the process of telomere clustering in meiotic prophase.…”

How plants LINC the SUN to KASH

“…1b). [34][35][36][37] This results in telomere clustering and later in nuclear oscillation between the cell poles, which facilitates homologous paring and recombination. 38 In mammals, a recent study revealed the formation of a Unknown MSP-300 D-Titin/Sallimus: anchoring mitochondria and endoplasmic reticulum to the striated muscle Z-discs.…”