Selection of axial growth sites in yeast requires Axl2p, a novel plasma membrane glycoprotein.

Abstract

Spa2p and Cdcl0p both participate in bud site selection and cell morphogenesis in yeast, and spa2~ cdclO-lO cells are inviable. To identify additional components important for these processes in yeast, a colony-sectoring assay was used to isolate high-copy suppressors of the spa2A cdclO-lO lethality. One such gene, AXL2, has been characterized in detail, axl2 cells are defective in bud site selection in haploid cells and bud in a bipolar fashion. Genetic analysis indicates that AXL2 falls into the same epistasis group as BUD3. Axl2p is predicted to be a type I transmembrane protein. Tunicamycin treatment experiments, biochemical fractionation and extraction experiments, and proteinase K protection experiments collectively indicate that Axl2p is an integral membrane glycoprotein at the plasma membrane. Indirect immunofluorescence experiments using either Axl2p tagged with three copies of a hemagglutinin epitope or high-copy AXL2 and anti-Axl2p antibodies reveal a unique localization pattern for Axl2p. The protein is present as a patch at the incipient bud site and in emerging buds, and at the bud periphery in small-budded cells. In cells containing medium-sized or large buds, Axl2p is located as a ring at the neck. Thus, Axl2p is a novel membrane protein critical for selecting proper growth sites in yeast. We suggest that Axl2p acts as an anchor in the plasma membrane that helps direct new growth components and/or polarity establishment components to the cortical axial budding site.

The Yck2 Yeast Casein Kinase 1 Isoform Shows Cell Cycle-specific Localization to Sites of Polarized Growth and Is Required for Proper Septin Organization

“…The Sec3 protein appears to be an exception, because it was reported to localize to a neck ring independent of septin function . Some of these proteins are also found at other areas of polarized growth, and this localization is not generally dependent on the septin ring (Adams and Pringle, 1984;Halme et al, 1996;Roemer et al, 1996). Similarly, Yck2p becomes concentrated in membranes of growing buds in the absence of septin function; however, we show here that association of the Yck2 kinase with the bud neck can be maintained in the absence of the septin ring.…”

“…The axial pattern of budding in haploid cells requires the action of the septin proteins (Flescher et al, 1993;, Bud3p and Bud4p , and the Axl1 and Axl2/Bud10 proteins (Fujita et al, 1994;Halme et al, 1996;Roemer et al, 1996). The diploid bipolar pattern is disturbed in mutants affecting the actin cytoskeleton (Schweitzer and Philippsen, 1991;Bauer et al, 1993;Amberg et al, 1997;Yang et al, 1997) as well as in Bud7p, Bud8p, Bud9p, Spa2p, and Bni1p (Zahner et al, 1996).…”

“…Most proteins required for polarized growth, cytokinesis, and bud site selection require the septin proteins for localization to the mother-bud neck Halme et al, 1996;Roemer et al, 1996;Sanders and Herskowitz, 1996;DeMarini et al, 1997;Lippincott and Li, 1998). One proposed function for the septin ring is to provide a structure on which assembly of other proteins can take place at appropriate times to promote bud site selection and to direct cytokinesis (Longtine et al, 1996).…”

Morphogenesis and the Cell Cycle

“…These define a machinery for properly placing and interpreting a set of guidepost or "landmark" proteins that are inherited by newborn cells at specific positions and influence subsequent bud placement. Many landmarks are integral plasma membrane proteins whose extracellular domains may interact with the cell wall to restrict their mobility, thereby preserving their initial localization (Halme et al 1996;Roemer et al 1996;Harkins et al 2001;Kang et al 2004a). The intracellular domains of the landmarks can interact with the GEF for the Ras-related Rsr1p GTPase (Kang et al 2001(Kang et al , 2004b, and this is thought to result in localized accumulation of GTP-Rsr1p near the landmark.…”

Transmembrane Topology of Pmt1p, a Member of an Evolutionarily Conserved Family of Protein O-Mannosyltransferases

“…Plasmid pSB52 (PMT1 G355HA )-A 111-bp NotI fragment encoding three copies of the hemagglutinin (HA) epitope was isolated from pAxl2 (22) and subcloned into pGEMEX-1 (Promega). Sequence analysis was used to identify clones with the 5Ј-sequence of the HA epitope following the XhoI site of the vector.…”