A new role for kinesin directed transport of Bik1p (CLIP-170) in Saccharomyces cerevisiae

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Microtubules (MTs) are fibrous structures in the cytoplasm of eukaryotic cells and play a vital role in cell organization, motility, and division. MTs are intrinsically polar polymers with a fast growing + end and a slow-growing - end (Carvalho et al., 2003; Desai and Mitchison, 1997). In fungi and animals, the - ends of MTs are usually at or adjacent to the microtubule organizing center and the + ends are oriented peripherally. A group of proteins called + end tracking proteins or +TIPs specifically associates with the + ends of MTs (Galjart and Perez, 2003). In live cell experiments, GFP-labelled + TIPs appear as comet- or dot-like structures that remain on the MT + ends (Perez et al., 1999). + TIPs form structural links between microtubule + ends and polarized membrane sites or kinetochores (Akhmanova et al., 2001; Carvalho et al., 2003; Coquelle et al., 2002; Dujardin and Vallee, 2002; Fukata et al., 2002; Howard and Hyman, 2003; Lin et al., 2001; Pierre et al., 1992; Tai et al., 2002) and are important for the regulation of microtubule dynamics (Han et al., 2001; Maiato et al., 2003; Rogers et al., 2002; Tirnauer et al., 1999).

The first discovered + TIP, CLIP-170, contains specific microtubule binding domains (CAP-Gly domains), which are shared by related proteins such as CLIP-115, P150^Glued, in mammalian cells, or its orthologs Bik1p and Tip1p in budding and fission yeasts. The CAP-Gly-containing proteins are central for astral microtubule interactions with cortical dynein-dynactin patches during mitosis and for the delivery of motors such as dynein to their site of action (Coquelle et al., 2002; Lansbergen et al., 2004; Niccoli et al., 2004; Tai et al., 2002; Vaughan et al., 2002; Xiang et al., 2000). In budding yeast, dynein is delivered to the cortex on the + ends of polymerizing MTs. Furthermore, dynein recruitment at MT tip is thought to involve dynein interactions with Bik1p at microtubule + ends (Sheeman et al., 2003).

How CLIP-170 and related proteins ‘recognize’ microtubule + ends is not completely clear. In mammalian cells, there is evidence that CLIP-170 accumulates at microtubule ends both by copolymerizing with tubulin (treadmilling mechanism) and by associating with another + TIP, EB1 (hitch hiking mechanism). An additional mechanism has been proposed in yeast where Bik1p and Tip1p are thought to be principally localized at microtubule + ends by kinesin motor proteins (Kip2p in budding yeast, Tea2p in fission yeast).

We have previously demonstrated that in budding yeast removal of the C-terminal phenylalanine residue of alpha tubulin dramatically impairs the recruitment of Bik1p at microtubule + ends (Badin-Larcon et al., 2004; Erck et al., 2005; Peris et al., 2006). We have recently reported related observations in mammalian cells, in which CLIP-170 and other CAP-Gly + TIPs association with microtubule + ends is severely impaired in cells expressing detyrosinated, Glu, tubulin. Several studies have provided a structural basis for our observations, by demonstrating a crucial requirement of the C-terminal aromatic residue of alpha-tubulin for tubulin interaction with CAP-Gly domains (Weisbrich et al., 2007)(+PNAS). In budding yeast; models in which Bik1p is principally localized as a Kip2p cargo do not require direct interactions of Bik1p with tubulin. There is an apparent conflict between such models and the massive mis-localization of Bik1p caused by the Glu mutation in budding yeast.

Here we demonstrate that, actually, whereas Kip2p can mediate the transport of Bik1p towards microtubule + ends, it cannot support subsequent Bik1p + end tracking. Additionally, our data indicate that dynein is transported at microtubule + ends in a complex with Kip2p and Bik1p. Apparently, dynein can subsequently track microtubule + ends even when Bik1p is severely depleted at those ends. Thus, although Kip2p cannot mediate Bik1p + end tracking as previously proposed, it seems to be central in a new pathway for dynein localization at + ends, which can support apparently normal dynein function even when the interaction of Bik1p with tubulin is disrupted.

In this study we provide strong evidence that Bik1p cannot end track microtubules as a cargo of Kip2p, as proposed in previous models. Our data indicate that although Bik1p is brought to microtubule + ends by Kip2p, microtubule end tracking by the two proteins are distinct processes. In yeast as in other systems, Bik1p seems to track + ends based on its intrinsic interaction with tubulin.

A key factor for deciphering the molecular interactions involved in Bik1p + end tracking has been the use of the Glu tubulin mutation, which, in recent studies, has been shown to specifically inhibit the interaction of CAP-Gly microtubule binding domains with the C-terminus of alpha tubulin. In our previous study, the Glu mutation only induced a three fold decrease of the Bik1p signal at microtubule ends. The existence of a sizeable remaining Bik1p signal could suggest incomplete inhibition of Bik1p interaction with tubulin by the Glu mutation in budding yeast. However, in this study, we demonstrate that in tub1-Glu bim1Δ strain the Bik1p signal at microtubule ends decreases to reach barely detectable levels. Such an effect of BIM1 deletion is compatible with the possibility that in Glu tubulin strains Bik1p localizes at + ends mainly by hitch-hiking Bim1p, not by interacting with Glu tubulin. Actually, in many cell types, Bik1p orthologs can be localize by hitch-hicking EB1 orthologs (Lansbergen and Akhmanova, 2006). Budding yeast looked as an exception, Bik1p localization being insensitive to BIM1 deletion in wt strains (Lin et al., 2001) (Fig. 1). Apparently the contribution of Bim1p hitch-hicking to Bik1p localization is minimal in wt strains, but becomes readily apparent in Glu tubulin mutant strains.

Our data indicate that Bik1p is transported to microtubule + ends in a complex with Kip2p which dissociates upon arrival at + ends with further microtubule + end tracking by Bik1p depending on Bik1p interaction with tubulin. What could trigger the dissociation the Bik1p/Kip2p complex at + ends? Possibly, Kip2p conformation and interactions with tubulin are different when the motor walks on microtubules and when it end-tracks microtubules. The two processes actually occur with profoundly different velocities, Kip2p motion on microtubules being 3 to 5 fold faster than microtubules growth (Carminati and Stearns, 1997; Carvalho et al., 2004; Shaw et al., 1997). In the case of another + end directed +TIP kinesin MCAK, + end tracking involves domains distinct from the motor domain, which is apparently inactive during end tracking (Moore et al., 2005). Kip2p may similarly be inactive as a motor during + end tracking, with a conformation which does not allow interaction with Bik1p. How could Bik1p end track microtubules, once dissociated from Kip2p? Possibly, Bik1p binds to free tubulin and co-polymerizes with tubulin exactly as suggested in the case of CLIP-170 in mammalian cells. However, such a model would only accounts for Bik1p tracking of growing + ends, whereas Bik1p also tracks shrinking + ends in budding yeast, suggesting the possibility of a direct association of Bik1p with + ends whether growing or shrinking; May be, the structures of growing and shrinking microtubule + ends, which are conspicuously different in the case of polymers made of mammalian tubulin (    ), are less different in polymers made from yeast tubulin.

Collectively, our data suggest that in wild type strains Bik1p tracks microtubule + ends principally based its intrinsic tubulin binding capacity, in a manner very much similar to that proposed for mammalian cells, and then functions to recruit dynein at microtubules + ends. Kip2p transport of Bik1p apparently facilitates Bik1p concentration close to + ends, without being strictly required. However, Kip2p is apparently central in a new functionally redundant pathway for Bik1p dependent dynein localization and transport. In this pathway, dynein is apparently brought to + ends in a ternary complex with Bik1p and Kip2p, and following dissociation of this complex, can end track microtubules even in the absence of Bik1p. Previous studies have indicated that dynein may normally end track microtubules in a ternary complex with Pac1p and Bik1p. May be, when Bik1p is lacking, dynein interaction with Pac1p is sufficient to sustain dynein tracking of + ends. One could then wonder why dynein could not be recruited directly by Pac1p. May be, dynein interaction with Bik1p releases an inhibition that otherwise precludes dynein interaction with Pac1p. A schematic summary of these models and conclusion is shown Figure 5.

Finally, it will be of obvious interest to know whether a kinesin dependent pathway for CLIP-170 mediated localization of dynein at + ends exists in mammalian cells. One way to know could be to test for the existence of dynein comets in tubulin tyrosine ligase deficient cells (Glu tubulin cells). The observation of such comets would strongly favour the possibility of kinesin mediated transport of both CLIP-170 and dynein in mammalian cells, as in yeast.