Loss-of-function mutations in seipin cause severe lipodystrophy, yet seipin's function in incompletely understood. Seipin is shown here to be important specifically for initiation of droplet formation, and a deletion mutant allows dissection of this function from maintenance of droplet morphology and vectorial droplet budding.

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In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.Telomeres are nucleoprotein structures that protect chromosome ends from nucleolytic digestion and preclude the recognition of normal ends as double-strand breaks (6). In most eukaryotes, telomeres are maintained by telomerase, a ribonucleoprotein that uses a short region of its RNA subunit as a template for reverse transcription. In Saccharomyces cerevisiae, a reverse transcriptase (RT) (EST2) and RNA subunit (TLC1) form the catalytic core of telomerase (18,21,35). Est2p contains at least three functional domains: an N-terminal TEN domain that anchors Est2p on its DNA substrate (23,24) and that may interact with other protein components (10), an RNA binding (RBD) region associates with that TLC1 RNA and contributes to dimerization (20), and an RT domain conserved among other telomerases and viral RTs (21).While many organisms synthesize perfect TG-rich telomeric repeats, several protozoa, fungi, slime molds, and plants have heterogeneous telomere sequences (40). S. cerevisiae displays considerable degeneracy, with a consensus of 5Ј-[(TG) 0-6 TG GGTGTG(G)] n (9). Several models have been proposed to explain this heterogeneity. An analysis of wild-type (WT) telomeres and telomeres generated in the presence of template mutations suggests that the registration of the telomere terminus occurs preferentially at the 3Ј end of the template, with processive synthesis through a central core region and decreasing processivity at the 5Ј end of the template (9, 33). In contrast, telomere junction fragments generated during chromosome healing events are more consistent with nonprocessive synthesis and patterning driven by substrate/template alignment (32). In humanized yeast cells, in which the yeast RNA template is replaced with that of humans, Est2p generates perfect hexanucleotide repeats, suggesting that the...

A loss of function of seipin, an endoplasmic reticulum (ER) membrane protein causes a severe congenital generalized lipodystrophy in humans. In yeast, deletion of seipin, FLD1, results in aberrant lipid droplets. However, the molecular mechanism of seipin is still unknown. Fei et al. (2011) reported that the deletion of FLD1 results in an increase in membrane phosphatidic acid (PA) concentration. In this study, we use Opi1p, a transcription repressor involved in phospholipid synthesis, as a PA sensor. Opi1p is known to bind to PA as well as the ER resident protein Scs2p. We report that Opi1p‐GFP forms puncta in the absence of FLD1. These puncta, usually next to droplets, are more pronounced with an Opi1p mutant (Opi1pFFAT) that cannot bind to Scs2p but still binds to PA. In order to test whether lipid droplets are necessary for the Opi1p‐GFP puncta in fld1Δ cells, we expressed Opi1p‐mCherry both in a mutant that cannot generate neutral lipids nor droplets (4KO; are1are2lro1dga1 KO), and one which also lacks FLD1 (5KO). Puncta were attenuated in these strains compared to fld1Δcells. In combination with the finding that Opi1p transiently localizes to droplets during inositol starvation (Chang et al, poster in this meeting), our data suggest that PA flux is important for droplet formation and that Fld1p facilitates utilization of PA during lipid droplet biogenesis. Supported by NIH grant GM084210.