Function of dynein in budding yeast: mitotic spindle positioning in a polarized cell.

Dyneins are AAA+ ATPases that use the energy from ATP hydrolysis to drive stepwise motility along microtubule tracks. Many dyneins are involved in axonemal function, found exclusively in organisms with cilia and flagella, but the cytoplasmic dynein 1 family is found in nearly all eukaryotes [Wickstead and Gull, 2007]. Cytoplasmic dynein is utilized for a variety of tasks, including transport of cargo along microtubules, positioning of the microtubule organizing center (MTOC), and organization of microtubule networks with respect to the cell cortex. The study of dynein in budding yeast began with the identification of the DYN1 (or DHC1) gene, which encodes the cytoplasmic dynein heavy chain [Eshel et al., 1993; Li et al., 1993]. The amino acid sequence of yeast Dyn1 is highly similar to that of the heavy chain of cytoplasmic dynein in metazoans (Fig. 1). The most conserved region is the carboxy-terminal motor domain. This region includes a ring of six AAA+ motifs plus a stalk domain that extends outward from the ring. The tip of this stalk contains the microtubule-binding domain of the motor [Carter et al., 2008]. ATP-binding and hydrolysis by the first and third of the AAA+ motifs is required for dynein function in cells, based on mutation of P loops; similar mutations of the other AAA+ motifs do not exhibit noticeable loss-of-dynein phenotypes [Reck-Peterson and Vale, 2004]. In vitro, the motor domain of Dyn1 alone is sufficient for motility in microtubule-gliding assays, suggesting that the function of the amino-terminal tail domain in cells may be to form complexes, bind cargo, or regulate dynein activity [Reck-Peterson et al., 2006]. Fig 1 Subunits of the dynein complex in Saccharomyces cerevisiae. Protein domain structure shown for dynein heavy chain, Dyn1, dynein intermediate chain, Pac11, dynein light intermediate chain, Dyn3, and dynein light chain (LC8-type), Dyn2. Dyn1 domain structure ... The only known function of dynein in budding yeast is to position the mitotic spindle during cell division. Whereas many eukaryotes employ dynein and microtubule-based mechanisms for the intracellular trafficking of organelles and other cargoes, budding yeast accomplish these processes primarily using the actin cytoskeleton, owing most likely to the small size of the yeast cell [Fagarasanu and Rachubinski, 2007]. Yeast undergo a closed mitosis, and therefore the nucleus and its mitotic spindle must be positioned across the junction between the mother and bud, termed the bud neck, to provide a set of chromosomes to the daughter cell. Null mutants of DYN1 often fail to move the spindle into the bud neck promptly, and cells proceed into anaphase with spindles and nuclei located entirely within the mother [Eshel et al., 1993; Li et al., 1993]. In most of these cases, the spindle does ultimately move into the neck, after a variable delay with cell-cycle arrest in late anaphase. The Kar9 pathway can compensate for the loss of dynein, using independent molecular mechanisms to move one end of the mitotic spindle into the nascent daughter cell. The Kar9 pathway guides cytoplasmic (i.e. astral) microtubules from one spindle pole body (SPB) into the bud by linking plus ends to a class-V myosin motor (Myo2) that moves along polarized cables of actin filaments [Miller and Rose, 1998; Hwang et al., 2003]. In addition, spindle elongation during anaphase B may help to push one pole into the bud. These compensatory processes require additional time to position the spindle, and this time is provided by a checkpoint mechanism that monitors the position of the spindle, delaying exit from mitosis until one SPB enters the bud [Adames et al., 2001; Molk et al., 2004]. Together, these mechanisms prevent the generation of bi-nucleate mothers and anucleate daughters. Many dynein components have been identified in yeast, owing to the viability and distinct phenotypes of dynein mutants. Genome-wide screens of haploid null mutants have identified a set of genes producing phenotypes similar to the loss of dynein, including nuclear segregation defects and synthetic genetic interactions with mutations in the Kar9 pathway [Tong et al., 2004; Lee et al., 2005; Li et al., 2005]. These dynein-pathway genes include homologues of many of the dynein constituents and regulators found throughout eukaryota. Divided into functional categories, these genes encode chains of the dynein motor (HC/Dyn1, IC/Pac11, LIC/Dyn3, LC8/Dyn2); the dynactin complex (p150glued/Nip100, p24/Yll049w, dynamitin/Jnm1, Arp1, Arp11/Arp10); the microtubule-associated proteins CLIP170/Bik1 and kinesin, Kip2; and the LIS1 and NudEL homologs, Pac1 and Ndl1. In addition, these screens have identified one gene without a clear functional homologue in higher eukaryotes — NUM1, which encodes a cortical protein.