Ageing and the mortality that ensues are sustainable for the species only if age is reset in newborns. In budding yeast, buds are made young whereas ageing factors, such as carbonylated proteins and DNA circles, remain confined to the ageing mother cell. The mechanisms of this confinement and their relevance are poorly understood. Here we show that a septin-dependent, lateral diffusion barrier forms in the nuclear envelope and limits the translocation of pre-existing nuclear pores into the bud. The retention of DNA circles within the mother cell depends on the presence of the diffusion barrier and on the anchorage of the circles to pores mediated by the nuclear basket. In accordance with the diffusion barrier ensuring the asymmetric segregation of nuclear age-determinants, the barrier mutant bud6Delta fails to properly reset age in buds. Our data involve septin-dependent diffusion barriers in the confinement of ageing factors to one daughter cell during asymmetric cell division.

In many cell types, lateral diffusion barriers compartmentalize the plasma membrane and, at least in budding yeast, the endoplasmic reticulum (ER). However, the molecular nature of these barriers, their mode of action and their cellular functions are unclear. Here, we show that misfolded proteins of the ER remain confined into the mother compartment of budding yeast cells. Confinement required the formation of a lateral diffusion barrier in the form of a distinct domain of the ER-membrane at the bud neck, in a septin-, Bud1 GTPase- and sphingolipid-dependent manner. The sphingolipids, but not Bud1, also contributed to barrier formation in the outer membrane of the dividing nucleus. Barrier-dependent confinement of ER stress into the mother cell promoted aging. Together, our data clarify the physical nature of lateral diffusion barriers in the ER and establish the role of such barriers in the asymmetric segregation of proteotoxic misfolded proteins during cell division and aging.DOI: http://dx.doi.org/10.7554/eLife.01883.001

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.

It is critical to elucidate the pathways that mediate spindle assembly and therefore ensure accurate chromosome segregation during cell division. Our studies of a unique allele of the budding yeast Ipl1/Aurora protein kinase revealed that it is required for centrosome-mediated spindle assembly in the absence of the BimC motor protein Cin8. In addition, we found that the Ase1 spindle midzone-associated protein is required for bipolar spindle assembly. The cin8 ipl1 and cin8 ase1 double mutant cells exhibit similar defects, and Ase1 overexpression completely restores spindle assembly in cin8 ipl1 strains. Consistent with the possibility that Ipl1 regulates Ase1, an ase1 mutant lacking the Ipl1 consensus phosphorylation sites cannot assemble spindles in the absence of Cin8. In addition, Ase1 phosphorylation and localization were altered in an ipl1 mutant. We therefore propose that Ipl1/Aurora and Ase1 constitute a previously unidentified spindle assembly pathway that becomes essential in the absence of Cin8.

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance.All eukaryotic cells are compartmentalized into distinct membrane-bound organelles and require tightly regulated transport of proteins and lipids between these compartments. Members of the Rab family of small GTPases are major regulators of protein and lipid traffic in the secretory and endocytic pathways (46,65). The action of Rab proteins in membrane transport was discovered with the identification of the SEC4 and YPT1 genes in yeast (Saccharomyces cerevisiae) (51, 52) and related proteins, termed Rab proteins, from mammals (10). Rab proteins comprise the most numerous subfamily of the Ras superfamily, and many are functionally uncharacterized. It is not clear whether Rab proteins regulate events that take place in the donor compartment, the vesicle or transport carrier, the acceptor compartment, or multiple locations. Also not known is whether a function(s) can be described for Rab proteins in general or whether this must be considered on a case-by-case basis. However, it is clear that Rab proteins are essential for eukaryotic cells and absolutely required for the function of all organelles connected by SNARE-mediated membrane traffic.With the completion of eukaryotic genome sequencing projects, there have been efforts to catalog the numbers of GTPase superfamily proteins. Membrane traffic in higher eukaryotes connects multiple compartments, and one reflection of this complexity may be the finding that humans have at least 60 different Rab family members (24,55,57). Other eukaryotes have fewer Rab proteins; Caenorhabditis elegans has 29 family members, Drosophila melanogaster has 26 members, and the yeast Saccharomyces cerevisiae has 11 members. As a model system, the 11 Rab proteins of the single-celled eukaryotic microbe S. cerevisiae can be considered the most minimal "membrome" (24), as other single-celle...

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