ribosome-free microcompartment between the two membranes. We found evidence for an Ist2-dependent modulation of Pma1 activity under specific conditions. Consistent with this Ist2 partially colocalized with the MCP. Results The transition from stationary into logarithmic growth phase requires Ist2 We characterized the growth of yeast cells lacking the IST2 gene. This mutant grew similarly to the isogenic wild type strain (WT) and both cultures reached a similar maximal density at stationary growth phase (Fig. 1A). However, the initial growth of ist2D was impaired during the adaptation from stationary phase to logarithmic (log) phase (Fig. 1A inset, indicated by arrows). During log phase the growth rate of ist2D accelerated (doubling time 177633 min) but remained slower than WT (11465 min). Comparing the growth of WT and ist2D, we saw a small but significant growth delay following dilution from a log culture (Fig. 1B, p,0.005). After dilution from late log-, transition-, and stationary phase cultures growth of ist2D cells was further delayed, when analyzed as growth compared to WT ten hours after dilution (54610%, 47614%, 48615%, p,0.001). Genomic integration of the IST2 gene into the his3D1 locus of ist2D cells rescued this Ist2 Mediates ER-Plasma Membrane Contacts adaptation defect. Western blot analysis of yeast membranes revealed a 4.861.6-fold higher expression of integrated IST2 as in WT (Fig. 1C). Expression of Ist2 from CEN plasmids led to similar expression as in WT (Fig. 1C and Fig. S1B) but did not rescue (Fig. 1B and Fig. S1A). Hence complementation by IST2 outside the genomic locus requires substantially higher Ist2 protein levels than present in the WT strain. This may be a consequence of IST2 mRNA transport into the bud [14,28] and a local function of Ist2. In order to analyze the phenotype of even stronger Ist2 overexpression we integrated the IST2 gene under control of the TEF1 promoter (pTEF1-IST2) into the his3D1 locus of WT cells. Analysis after serial dilution of membranes from pTEF1-IST2 revealed 25-fold higher expression as in WT (Fig. 1D). Compared to the isogenic WT the pTEF1-IST2 strain showed a delayed growth after dilution from a stationary culture (Fig. 1E). As seen for ist2D the growth of pTEF1-IST2 accelerated during log phase with a doubling time of 178613 min (Fig. S1C). Taken together these data suggest that the amount of Ist2 protein is critical for the transition from stationary to log growth. Ist2 recruits yeast cortical ER to the PM Ist2 is a polytopic membrane protein that contains a lipid- binding domain that recognizes PI(4,5)P2 and other lipids at the Figure 1. Ist2 is required for the transition from stationary to log phase. (A and B) Growth curves of WT and ist2D strains in complete synthetic media (HC). The cultures were diluted to 0.05 OD600 from stationary cultures (OD600.3.0) and incubated at 25uC. Curves in A are semi-log, in B linear showing an enlargement of the initial growth. The arrow indicates the difference between WT and ist2D after 10 hours. Doubling time for log phase growth was calculated between 0.4 and 0.8 OD600. (C) Growth of ist2D or WT cells transformed with an empty plasmid (pRS303) and of ist2D with either IST2 integrated into the HIS3 locus (ist2D+IST2) or with IST2 encoded by a CEN, HIS3 plasmid (ist2D+IST2/CEN). Cultures were diluted to OD600 0.05 from pre-cultures with an OD600 1.0–2.0 (log), OD600 2.0–2.5 (late log), OD600 2.5–3.0 (transition), and OD600.3.0 (stationary). Growth relative to WT (set to 100%) after 10 hours is shown. Error bars depict s.d., non-significant differences are indicated as n.s., and triple asterisks show significance (p,0.001). (D) Membranes prepared from 5 OD600 of the strains described in C were separated. Ist2 and Sec61 were detected with specific antibodies. The amounts of Ist2 were quantified in membranes from three independent preparations and the s.d. is shown. (E) Relative growth of WT (set to 100%) and pTEF1-IST2 strains 10 hours after dilution from stationary pre-cultures. Error bars depict s.d. doi:10.1371/journal.pone.0039703.g001 PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e39703