AIP3/BUD6

Aip3p is a 98 kDa protein that was originally discovered during Dr. Amberg's post-doc as a protein that interacted with yeast actin in a two-hybrid assay. Approximately the same time, Joe Zahner, working in John Pringle's lab identified a mutant allele of aip3 (they called it BUD6) Aip3Localethat caused random bud site selection in yeast diploids. At this time, the BUD6 moniker has prevailed and so this gene is commonly known as BUD6 not AIP3. Besides defects in dipoid bud site selection, bud6? celles have severe defects in actin polarization, in particular the formation of polarized actin cables which are so important for the polarized deliver of secretory vesicles and organelle inheritance. Our preliminary analysis of Aip3p/Bud6p was jointly published with the Pringle lab:

Amberg DC, Zahner JE, Mulholland, Pringle JR and Botstein D. Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites. (1997) Mol. Biol. Cell; 8: 729-753.

Bud6p displays a localization pattern that is typical for proteins that regulate actin and cell polarization in yeast. At the beginning of the cell cycle it localizes to the site of bud emergence, then it remains at the bud cortex until later in the cell cycle when it moves to form two rings on either side of the neck. At right is shown the localization of GFP-Bud6p expressed in diploid yeast cells.

GFPBud6p

Initially, we became interested in the regulation of Bud6p localization as a means by which to get a handle on how the cell cycle controls the localization of polarity regulators. Surprisingly, initial fractionation studies suggested that a pool of Bud6p is associated with membrane structures and careful follow up studies strongly argued that Bud6p is associated with secretory vesicles. These biochemical studies were supported by our observations that Bud6p fails to polarize in secretory pathway mutants and instead accumulates on aberrant intra-cellular membranes. We mapped the region of Bud6p that was sufficient for secretory pathway mediated delivery of the protein to the cell surface to the N-terminal 1/3 of the protein. This work was important as it provided the first molecular explanation for the apparent coupling of secretion to actin polarization; Bud6p is an important determinant in the establishment of actin polarization and the subsequent deliver of Bud6p on secretory vesicles functions to reinforce actin polarization in an auto-feedback mechanism. These studies were published in the following manuscript:

Jin H and Amberg DC. The secretory pathway mediates localization of the cell polarity regulator Aip3p/Bud6p. (2000) Molec. Biol. Cell; 11: 647-661.

spBud6 LocaleThis study was followed by a brief foray into S. pombe biology when we became interested in whether the fission yeast homolog of Bud6 performed similar function in this highly divergent yeast. We confirmed that pombe Bud6 also interacts with actin and can localize and function when expressed in S. cerevisiae. Regulation of S. pombe polarity is rather different than the regulation of S. cerevisiae cell polarity in that polarity is directed by the microtubule cytoskeleton and the actin cytoskeleton is subservient to the microtubules.

Therefore, it was not so surprising that bud6 deletion in S. pombe had little effect on polarized growth. However, when we crippled the mictotubules with a microtubule de-polymerizing drug, spBud6p became important for the establishment of actin polarization and polarized cell growth. As for budding yeast, in S. pombe Bud6p localization was completely dependent on the secretory pathway however, we showed that this depended on microtubules for GFPspBud6pdelivery of Bud6p to the cell ends and on actin for deliver of Bud6 to the cell middle. Finally, we showed that Bud6p over-expression caused severe defects in S. pombe cell polarity and led Bud6p to localize to microtubules, the first evidence that Bud6p might be a microtubule binding protein. These experiments were published in the following article:

Jin H and Amberg DC. (2001) Fission yeast Aip3p (spAip3p) is required for an alternative actin-directed polarity program. (2001) Molec. Biol. Cell; 12: 1275-1291.

In the subsequent years, several laboratories have made additional important discoveries concerning the functions of Bud6p, which has turned out to be a very functionally diverse protein. Charlie Boone's lab first showed that Bud6p interacts with the formin Bni1p and the Snyder lab reinforced this observation by showing that Bud6p is in a complex they call the polarisome which includes the scaffold protein Spa2p as well as Bni1p and Pea2p. Biochemical analyses of Bud6p done in the Goode lab have very beautifully shown that the C-terminus of Bud6p dramatically activates the normally wimpy actin nucleation activity of Bni1p. These experiments explain an old observation of ours that bni1? and bud6? strains have equally serious defects in actin polarization and that the double mutant is nor worse than either single mutant.

Interestingly, Bud6p appears to have a separate function in regulating nuclear positioning during mitosis. Marisa Segal's lab has done an elegant series of experiments showing that astral microtubules are captured at cortical sites of Bud6p localization and that this is part of a system that draws the dividing nucleus into the neck region in preparation for anaphase. Quite recently, we have reported that Bud6p is also localized to the spindle pole body and is part of a retention system that holds the dividing nucleus in the neck just prior to the onset of anaphase. Two movies are shown below both of cells expressing a truncated non-functional form of Bud6p that localizes both to the spindle pole body and the neck. The first movie shows pre-anaphase nuclear movements in a wild type cell while the second movie shows pre-anaphase nuclear movements in a bud6? strain. As one can see, in the wild type cells the spindle pole bodies are never drawn past the middle of the neck while in the bud6? cells the nucleus swings wildly through the neck. Also notice that there is an apparently stabilizing contact between the spindle pole body and the neck. There still exists a lot of controversy over how the cell knows when to initiate anaphase. We think these most recent studies from our lab suggest that the signal for completion of the cell cycle may involve contact/residence time of the spindle pole body on the neck. This study was just accepted for publication in Eukaryotic Cell:

Haarer BK, Helfant AH, and Amberg DC. Stable pre-anaphase spindle positioning requiresBud6p/Aip3p and an apparent interaction betw een the spindle pole bodies and the neck. (2007) Euk. Cell, May 2007; 6 (5): 797-807

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