Cytoplasmic dynein is required for normal nuclear segregation in yeast.

Abstract

We have identified the gene DYNI, which encodes the heavy chain of cytoplasmic dynein in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence (Mr 471,305) reveals the presence of four P-loop motifs, as in all dyneins known so far, and has 28% overall identity to the dynein heavy chain ofDictyostelium [Koonce, M. P., Grissom, P. M. & McIntosh, J. R. (1992) J. CeUl Biol. 119, 1597Biol. 119, -1604 with 40% identity in the putative motor domain. Disruption of DYNI causes misalignment of the spindle relative to the bud neck during cell division and results in abnormal distribution of the dividing nuclei between the mother cell and the bud. Cytoplasmic dynein, by generating force along cytoplasmic microtubules, may play an important role in the proper alignment of the mitotic spindle in yeast.Nuclear and cytoplasmic microtubules in the yeast Saccharomyces cerevisiae participate in several well-defined cellular processes that include the segregation of chromosomes and the migration of the nucleus during mitosis as well as the migration and fusion of nuclei in karyogamy (1). These processes are believed to involve microtubule-based motor enzymes, and recently several genes containing sequence regions with homology to the motor domain characteristic of the kinesin superfamily have been identified in S. cerevisiae and other fungi (2-8).The cytoplasmic form ofthe microtubule-associated motor dynein has ATPase activity and moves toward the minus ends of microtubules in in vitro gliding assays (9). Cytoplasmic dynein exists in a wide variety of eukaryotic cells, including Dictyostelium (10, 11), and indirect evidence has suggested that it plays a major role in the movement of chromosomes toward the minus ends of spindle microtubules at the anaphase stage of mitosis (12, 13), as well as in the retrograde transport of organelles in nerve axons (14)(15)(16) Transformations were done with the lithium acetate method (18). Synchronization of yeast cultures was achieved by treating with a mating factor as described by Berlin et al (19).PCR Primers. Oligonucleotide primers were designed from the deduced amino acid sequence of the sea urchin dynein ,B heavy chain (20, 21) in regions that were conserved between sea urchin dynein isoforms (22). The nucleotide sequences of the two degenerate primers that were used to PCR-amplify pDLP1 from yeast genomic DNA are 5'-CCTGCTGGNAC-NGGNAARAC-3' (sense strand, targeting amino acidsequence PAGTGKT) and 5'-TACCCIGGRTTCATIGTDA-TRAA-3' (antisense strand, targeting amino acid-sequence FITMNPG).DNA Sequencing. Nucleotide sequencing of the original probe, pDLP1, and other restriction fragments in the vicinity of the putative hydrolytic ATP-binding site was done by subcloning into M13mpl8/mp19 vectors and using universal M13 primers and a Sequenase 2.0 DNA sequencing kit (United States Biochemical).The nucleotide sequence of the complete gene was determined as part of the ongoing project to sequence the entire chromosome XI in yeast (strain S288C). Relevant cosmids were soni...

The dynein genes of Paramecium tetraurelia: the structure and expression of the ciliary beta and cytoplasmic heavy chains.

“…There is a clear separation between the three 13 chains and the nine cytoplasmic dyneins (bold font). Dynein heavy chain sequences and their abbreviations used in this table are as follows: DHC-6, Paramecium 13 (this study; accession number U19464); SU 13, sea urchin 13 X59603); Chlamy 13, Chlamydomonas 13 (Mitchell and Brown, 1994; U02963); Chlamy -y, Chlamydomonas y (Wilkerson et al, 1994); DHC-8, Paramecium cytoplasmic (this study; U20449); Dicty, Dictyostelium (Koonce et al, 1992;Z15124); Dro cyto, Drosophila cytoplasmic (Li et al, 1994;L23195); MAPlC, rat cytoplasmic (Mikami et al, 1993;L08505); C eleg, Caenorhabditis rhabditis (Lye et al, 1995;L33260); SU cyto, sea urchin la Z21941); N crassa, Neurospora crassa (Plamann et al, 1994;L31504); Asp, Aspergillus nidulans (Xiang et al, 1994;U03904); Yeast, Saccharomyces cerevisiae (Eshel et al, 1993;Z21877 Ogawa, 1991), Chlamydomonas (Mitchell and Brown, 1994), and Paramecium (this report); the axonemal y heavy chain from Chlamydomonas (Wilkerson et al, 1994); and the cytoplasmic dyneins from Dictyostelium (Koonce et al, 1992), rat brain (Mikami et al, 1993;Zhang et al, 1993), S. cerevisiae (Eshel et al, 1993;Li et al, 1993), Aspergillus (Xiang et al, 1994), Neurospora (Plamann et al, 1994), Drosophila (Li et al, 1994), C. elegans (Lye et al, 1995), and Paramecium (this report cated by potential differences due to species and tissue variation.…”

Functional Coordination of Three Mitotic Motors inDrosophilaEmbryos

“…First, we show that cytoplasmic dynein is involved in positioning spindle poles throughout all stages of spindle assembly and elongation; because dynein is not necessary for proper spindle pole separation before anaphase in fungi (Eshel et al, 1993;Li et al, 1993;Xiang et al, 1994;Yeh et al, 1995), its role(s) in the early stages of mitosis could not be examined. Second, we show that the Drosophila C-terminal kinesin Ncd constrains the rate of spindle pole separation during spindle Figure 8.…”

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

“…Yeast have two partially redundant nuclear positioning pathways: the dynein- and Kar9-mediated pathways [2,25,26]. In the absence of both pathways, cells exhibit a severe growth defect or are inviable.…”

“…We also examined spindle orientation as a readout for dynein function in PAN cells (Figure 3(b)) [2]. Cells expressing Num1ΔPH exhibited an increase in spindle misorientation similar to Δ dyn1 and Δ num1 cells (Figure 3(c)).…”