Biomedical subjects

Susan Ferro-Novick

Publications and source records attributed to Susan Ferro-Novick.

13 recordsLinked to original sources

PubMed2006

mBET3 is required for the organization of the TRAPP complexes.

Large tethering complexes mediate the initial interaction of a transport vesicle with its target membrane. There are two forms of the multi-subunit tethering complex called TRAPP (TRAPPI and TRAPPII) that tether transport vesicles in different trafficking steps. Understanding TRAPP complex assembly and the protein-protein interactions among the subunits is an important step in elucidating the function of this tether. Here we have used several different approaches to study the protein-protein interactions among the subunits of the TRAPP complexes in both mammalian cells and yeast. Our studies have revealed that the low molecular weight subunits of TRAPP form two subcomplexes in vitro. One subcomplex contains mammalian BET3 (mBET3), mTRS31 and mTRS20, while mBET5 and mTRS23 form a second subcomplex. Furthermore, mBET3 directly interacts with mBET5 in vitro. Our findings also suggest that the TRAPPII-specific subunit, yTrs120p (yeast Trs120p), binds to the periphery of the TRAPPII complex. Although the non-essential TRAPP subunit yTrs33p interacts with yBet3p, yTrs33p is not required for TRAPP complex assembly. Together our findings indicate that BET3 plays an important role in the organization of the TRAPP complexes in both mammalian cells and yeast.

Animals↗

PubMed2006

Ptc1p regulates cortical ER inheritance via Slt2p.

Studies in the yeast Saccharomyces cerevisiae have shown that the inheritance of endoplasmic reticulum (ER), mitochondria, and vacuoles involves the capture of a tubular structure at the bud tip. Ptc1p, a serine/threonine phosphatase, has previously been shown to regulate mitochondrial inheritance by an unknown mechanism. Ptc1p regulates the high osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway and has also been implicated in the cell wall integrity (CWI) MAPK pathway. Here we show that the loss of Ptc1p or the Ptc1p binding protein, Nbp2p, causes a prominent delay in the delivery of ER tubules to the periphery of daughter cells and results in a dramatic increase in the level of phosphorylated Slt2p, the MAPK in the CWI pathway. Either loss of Slt2p or inhibition of the CWI pathway by addition of sorbitol, suppresses the ER inheritance defect in the ptc1Delta and nbp2Delta mutants. Our findings indicate that Ptc1p and Nbp2p regulate ER inheritance through the CWI MAPK pathway by modulating the MAPK, Slt2p.

Adaptor Proteins, Signal Transducing↗

PubMed2006

mBet3p is required for homotypic COPII vesicle tethering in mammalian cells.

TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.

Animals↗

PubMed2006

Rtn1p is involved in structuring the cortical endoplasmic reticulum.

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Delta in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Delta cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

Endoplasmic Reticulum↗

PubMed2005

Mutants in trs120 disrupt traffic from the early endosome to the late Golgi.

Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.

Coat Protein Complex I↗

PubMed2005

The organization, structure, and inheritance of the ER in higher and lower eukaryotes.

The endoplasmic reticulum (ER) is a fundamental organelle required for protein assembly, lipid biosynthesis, and vesicular traffic, as well as calcium storage and the controlled release of calcium from the ER lumen into the cytosol. Membranes functionally linked to the ER by vesicle-mediated transport, such as the Golgi complex, endosomes, vacuoles-lysosomes, secretory vesicles, and the plasma membrane, originate largely from proteins and lipids synthesized in the ER. In this review we will discuss the structural organization of the ER and its inheritance.

Animals↗

PubMed2004

Ice2p is important for the distribution and structure of the cortical ER network in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found along the cell periphery (cortical ER) and nucleus (perinuclear ER). In this study, we characterize a novel ER protein called Ice2p that localizes to the cortical and perinuclear ER. Ice2p is predicted to be a type-III transmembrane protein. Cells carrying a genomic disruption of ICE2 display defects in the distribution of cortical ER in mother and daughter cells. Furthermore, fluorescence imaging of ice2delta cells reveals an abnormal cortical ER tubular network morphology in both the mother cell and the developing bud. Subcellular fractionation analysis using sucrose gradients corroborate the data from the fluorescence studies. Our findings indicate that Ice2p plays a role in forming and/or maintaining the cortical ER network in budding yeast.

Endoplasmic Reticulum↗

PubMed2004

Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p.

The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Delta, sec5Delta, and exo70Delta strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.

Cell Division↗

PubMed2004

Dynamics and inheritance of the endoplasmic reticulum.

The endoplasmic reticulum (ER) consists of a polygonal array of interconnected tubules and sheets that spreads throughout the eukaryotic cell and is contiguous with the nuclear envelope. This elaborate structure is created and maintained by a constant remodeling process that involves the formation of new tubules, their cytoskeletal transport and homotypic fusion. Since the ER is a large, single-copy organelle, it must be actively segregated into daughter cells during cell division. Recent analysis in budding yeast indicates that ER inheritance involves the polarized transport of cytoplasmic ER tubules into newly formed buds along actin cables by a type V myosin. The tubules then become anchored to a site at the bud tip and this requires the Sec3p subunit of the exocyst complex. The ER is then propagated along the cortex of the bud to yield a cortical ER structure similar to that of the mother cell. In animal cells, the ER moves predominantly along microtubules, whereas actin fibers serve a complementary role. It is not yet clear to what extent the other components controlling ER distribution in yeast might be conserved in animal cells.

Actins↗

PubMed2003

Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae.

Myo4p is a nonessential type V myosin required for the bud tip localization of ASH1 and IST2 mRNA. These mRNAs associate with Myo4p via the She2p and She3p proteins. She3p is an adaptor protein that links Myo4p to its cargo. She2p binds to ASH1 and IST2 mRNA, while She3p binds to both She2p and Myo4p. Here we show that Myo4p and She3p, but not She2p, are required for the inheritance of cortical ER in the budding yeast Saccharomyces cerevisiae. Consistent with this observation, we find that cortical ER inheritance is independent of mRNA transport. Cortical ER is a dynamic network that forms cytoplasmic tubular connections to the nuclear envelope. ER tubules failed to grow when actin polymerization was blocked with the drug latrunculin A (Lat-A). Additionally, a reduction in the number of cytoplasmic ER tubules was observed in Lat-A-treated and myo4Delta cells. Our results suggest that Myo4p and She3p facilitate the growth and orientation of ER tubules.

Actin Cytoskeleton↗

PubMed2003

Sec3p is needed for the spatial regulation of secretion and for the inheritance of the cortical endoplasmic reticulum.

Sec3p is a component of the exocyst complex that tethers secretory vesicles to the plasma membrane at exocytic sites in preparation for fusion. Unlike all other exocyst structural genes, SEC3 is not essential for growth. Cells lacking Sec3p grow and secrete surprisingly well at 25 degrees C; however, late markers of secretion, such as the vesicle marker Sec4p and the exocyst subunit Sec8p, localize more diffusely within the bud. Furthermore, sec3Delta cells are strikingly round relative to wild-type cells and are unable to form pointed mating projections in response to alpha factor. These phenotypes support the proposed role of Sec3p as a spatial landmark for secretion. We also find that cells lacking Sec3p exhibit a dramatic defect in the inheritance of cortical ER into the bud, whereas the inheritance of mitochondria and Golgi is unaffected. Overexpression of Sec3p results in a prominent patch of the endoplasmic reticulum (ER) marker Sec61p-GFP at the bud tip. Cortical ER inheritance in yeast has been suggested to involve the capture of ER tubules at the bud tip. Sec3p may act in this process as a spatial landmark for cortical ER inheritance.

Carrier Proteins↗

PubMed2003

The Yip1p.Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles.

Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p.Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

A Ypt32p exchange factor is a putative effector of Ypt1p.

Ypt1p regulates vesicle tethering and fusion events from the ER to the Golgi and through the early Golgi. Genetic studies have suggested a functional relationship between Ypt1p and Ypt31p/Ypt32p. Ypt31p and Ypt32p are a pair of functionally redundant GTPases that act after Ypt1p to mediate intra-Golgi traffic or the budding of post-Golgi vesicles from the trans-Golgi. Here we report that a novel Ypt32p exchange factor is a putative effector of Ypt1p. These findings implicate small GTP-binding proteins of the Ypt/Rab family in a signal cascade that directs membrane traffic through the secretory pathway.

Carrier Proteins↗