exhibiting elongated MCC patches, there are elongated invaginations of the appropriate size and frequency. Using various approaches of immunoelectron microscopy, the MCC protein Sur7, as well as the eisosome marker Pil1, have been detected at these invaginations. Thus, we identify the MCC patch, which is a lateral membrane domain of specific composition and function, with a specific structure in the yeast plasma membrane -the furrow-like invagination.

The plasma membrane of the yeast Saccharomyces cerevisiae contains stably distributed lateral domains of specific composition and structure, termed MCC (membrane compartment of arginine permease Can1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an NCE102 deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the NCE102 close homolog FHN1 (the previously uncharacterized YGR131W) as well as by distant Schizosaccharomyces pombe homolog fhn1 (SPBC1685.13). We conclude that this mechanism of plasma membrane organization is conserved through the phylum Ascomycota. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.Stable lateral domains coexist within the plasma membrane of the yeast Saccharomyces cerevisiae. Nce102, a protein originally thought to be involved in nonclassical export (6) and more recently in sensing sphingolipids (10), is the main organizer of one type of these domains, termed MCC (membrane compartment of Can1) (25). MCC consists of evenly distributed, isolated patches enriched in sterols and specific proteins (15,16,25,26). We showed that MCC-specific proton symporters accumulate in these patches in a reversible, membrane potential-dependent manner. This Nce102-mediated transient MCC accumulation plays a key role in the turnover of the transporters (16). Each MCC patch is accompanied by an eisosome, a cytosolic complex located directly beneath the membrane (36).In an early freeze-etching study, Moor and Mühlethaler (28) demonstrated that the yeast plasma membrane contains numerous furrow-like invaginations. Recently, MCC patches were identified with these plasma membrane structures, and Nce102 was shown to be necessary for furrow formation. On the ultrastructural level, the MCC patches of nce102⌬ cells appeared as flat, smooth, elongated areas within an otherwise particle-rich plasma membrane (32).There is now increasing evidence that cytosolic Pil1, a primary component of eisosomes, is a prerequisite for MCC patch formation. It marks the sites where Nce102 and the MCCspecific transporters will subsequently accumulate (16,23,29). Data published so far do not indicate a direct involvement of cytoskeletal components in this process (26). Accordingly, markers of classical endocytosis, which are coupled to the cortical patches of actin, were localized outside the MCC ...

In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis.

Lipid droplets are found in most organisms where they serve to store energy in the form of neutral lipids. They are formed at the endoplasmic reticulum (ER) membrane where the neutral-lipidsynthesizing enzymes are located. Recent results indicate that lipid droplets remain functionally connected to the ER membrane in yeast and mammalian cells to allow the exchange of both lipids and integral membrane proteins between the two compartments. The precise nature of the interface between the ER membrane and lipid droplets, however, is still ill-defined. Here, we probe the topology of lipid droplet biogenesis by artificially targeting proteins that have high affinity for lipid droplets to inside the luminal compartment of the ER. Unexpectedly, these proteins still localize to lipid droplets in both yeast and mammalian cells, indicating that lipid droplets are accessible from within the ER lumen. These data are consistent with a model in which lipid droplets form a specialized domain in the ER membrane that is accessible from both the cytosolic and the ER luminal side.

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/ topogenesis of mitochondrial outer membrane b-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-b indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs. monomers and state-3/ state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMMdetached cristae of OPA1-and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-

Regulation of gene expression on the level of translation and mRNA turnover is widely conserved evolutionarily. We have found that the main mRNA decay enzyme, exoribonuclease Xrn1, accumulates at the plasma membrane-associated eisosomes after glucose exhaustion in a culture of the yeast S. cerevisiae. Eisosomal localization of Xrn1 is not achieved in cells lacking the main component of eisosomes, Pil1, or Sur7, the protein accumulating at the membrane compartment of Can1 (MCC) - the eisosome-organized plasma membrane microdomain. In contrast to the conditions of diauxic shift, when Xrn1 accumulates in processing bodies (P-bodies), or acute heat stress, in which these cytosolic accumulations of Xrn1 associate with eIF3a/Rpg1-containing stress granules, Xrn1 is not accompanied by other mRNA-decay machinery components when it accumulates at eisosomes in post-diauxic cells. It is important that Xrn1 is released from eisosomes after addition of fermentable substrate. We suggest that this spatial segregation of Xrn1 from the rest of the mRNA-decay machinery reflects a general regulatory mechanism, in which the key enzyme is kept separate from the rest of mRNA decay factors in resting cells but ready for immediate use when fermentable nutrients emerge and appropriate metabolism reprogramming is required. In particular, the localization of Xrn1 to the eisosome, together with previously published data, accents the relevance of this plasma membrane-associated compartment as a multipotent regulatory site.

As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells.

A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0 degrees C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.