Biomedical subjects

Elizabeth Sztul

Publications and source records attributed to Elizabeth Sztul.

At least 19 recordsLinked to original sources

PubMed2006

SNARE status regulates tether recruitment and function in homotypic COPII vesicle fusion.

In mammals, coat complex II (COPII)-coated transport vesicles deliver secretory cargo to vesicular tubular clusters (VTCs) that facilitate cargo sorting and transport to the Golgi. We documented in vitro tethering and SNARE-dependent homotypic fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers characteristic of VTCs ( Xu, D., and Hay, J. C. (2004) J. Cell Biol. 167, 997-1003). COPII vesicles thus appear to contain all necessary components for homotypic tethering and fusion, providing a pathway for de novo VTC biogenesis. Here we demonstrate that antibodies against the endoplasmic reticulum/Golgi SNARE Syntaxin 5 inhibit COPII vesicle homotypic tethering as well as fusion, implying an unanticipated role for SNAREs upstream of fusion. Inhibition of SNARE complex access and/or disassembly with dominant-negative alpha-soluble NSF attachment protein (SNAP) also inhibited tethering, implicating SNARE status as a critical determinant in COPII vesicle tethering. The tethering-defective vesicles generated in the presence of dominant-negative alpha-SNAP specifically lacked the Rab1 effectors p115 and GM130 but not other peripheral membrane proteins. Furthermore, Rab effectors, including p115, were shown to be required for homotypic COPII vesicle tethering. Thus, our results demonstrate a requirement for SNARE-dependent tether recruitment and function in COPII vesicle fusion. We anticipate that recruitment of tether molecules by an upstream SNARE signal ensures that tethering events are initiated only at focal sites containing appropriately poised fusion machinery.

Amino Acid Sequence↗

PubMed2006

Oocyte signals derived from polyunsaturated fatty acids control sperm recruitment in vivo.

A fundamental question in animal development is how motile cells find their correct target destinations. During mating in the nematode Caenorhabditis elegans, males inject sperm through the hermaphrodite vulva into the uterus. Amoeboid sperm crawl around fertilized eggs to the spermatheca--a convoluted tube where fertilization occurs. Here, we show that polyunsaturated fatty acids (PUFAs), the precursors of eicosanoid signalling molecules, function in oocytes to control directional sperm motility within the uterus. PUFAs are transported from the intestine, the site of fat metabolism, to the oocytes yolk, which is a lipoprotein complex. Loss of the RME-2 low-density lipoprotein (LDL) receptor, which mediates yolk endocytosis and fatty acid transport into oocytes, causes severe defects in sperm targeting. We used an RNAi screen to identify lipid regulators required for directional sperm motility. Our results support the hypothesis that PUFAs function in oocytes as precursors of signals that control sperm recruitment to the spermatheca. A common property of PUFAs in mammals and C. elegans is that these fats control local recruitment of motile cells to their target tissues.

Animals↗

PubMed2006

On and off membrane dynamics of the endoplasmic reticulum-golgi tethering factor p115 in vivo.

The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t1/2 approximately 20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t1/2 approximately 13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP. p115 mutant missing the GM130/giantin binding site exhibits analogous fluorescence recovery after photobleaching (FRAP) (t1/2 approximately 13 s), suggesting that GM130 and giantin are not major determinants of p115 membrane dynamics. In contrast, p115-GFP exchanges more rapidly (t1/2 approximately 8 s) in cells expressing the inactive Rab1/N121I mutant, indicating that p115 cycling is influenced by Rab1. p115-GFP dynamics is also influenced by the assembly status of SNAREs. In cells expressing an ATPase-deficient NSF/E329Q mutant that inhibits SNARE complex disassembly, the cycling kinetics of p115-GFP are significantly slower (t1/2 approximately 21 s). In contrast, in cells incubated at reduced temperature (10 degrees C) that inhibits vesicular traffic, the cycling kinetics of p115-GFP are faster (t1/2 approximately 7 s). These data suggest that p115-binding sites on the membrane are provided by unassembled SNAREs. In agreement, biochemical studies show increased p115 recruitment to membranes in the presence of NSF and alpha-SNAP. Our data support a model in which recruitment of tethers is directly regulated by the assembly status of SNAREs.

Animals↗

PubMed2006

Role of tethering factors in secretory membrane traffic.

Coiled-coil and multisubunit tethers have emerged as key regulators of membrane traffic and organellar architecture. The restricted subcellular localization of tethers and their ability to interact with Rabs and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) suggests that tethers participate in determining the specificity of membrane fusion. An accepted model of tether function considers them molecular "bridges" that link opposing membranes before SNARE pairing. This model has been extended by findings in various experimental systems, suggesting that tethers may have other functions. Recent reports implicate tethers in the assembly of SNARE complexes, cargo selection and transit, cytoskeletal events, and localized attachment of regulatory proteins. A concept of tethers as scaffolding machines that recruit protein components involved in varied cellular responses is emerging. In this model, tethers function as integration switches that simultaneously transmit information to coordinate distinct processes required for membrane traffic.

Animals↗

PubMed2005

Nuclear aggresomes form by fusion of PML-associated aggregates.

Nuclear aggregates formed by proteins containing expanded poly-glutamine (poly-Q) tracts have been linked to the pathogenesis of poly-Q neurodegenerative diseases. Here, we show that a protein (GFP170*) lacking poly-Q tracts forms nuclear aggregates that share characteristics of poly-Q aggregates. GFP170* aggregates recruit cellular chaperones and proteasomes, and alter the organization of nuclear domains containing the promyelocytic leukemia (PML) protein. These results suggest that the formation of nuclear aggregates and their effects on nuclear architecture are not specific to poly-Q proteins. Using GFP170* as a model substrate, we explored the mechanistic details of nuclear aggregate formation. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses show that GFP170* molecules exchange rapidly between aggregates and a soluble pool of GFP170*, indicating that the aggregates are dynamic accumulations of GFP170*. The formation of cytoplasmic and nuclear GFP170* aggregates is microtubule-dependent. We show that within the nucleus, GFP170* initially deposits in small aggregates at or adjacent to PML bodies. Time-lapse imaging of live cells shows that small aggregates move toward each other and fuse to form larger aggregates. The coalescence of the aggregates is accompanied by spatial rearrangements of the PML bodies. Significantly, we find that the larger nuclear aggregates have complex internal substructures that reposition extensively during fusion of the aggregates. These studies suggest that nuclear aggregates may be viewed as dynamic multidomain inclusions that continuously remodel their components.

Aggrecans↗

PubMed2005

Transcriptional repression and cell death induced by nuclear aggregates of non-polyglutamine protein.

Nuclear aggregates of polyglutamine (polyQ)-expanded proteins are associated with a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxias (SCAs). The nuclear deposition of polyQ proteins correlates with rearrangements of nuclear matrix, transcriptional dysregulation, and cell death. To explore the requirement for polyQ tracks in educing such cellular responses, we examined whether a non-polyQ protein can deposit as nuclear aggregates and elicit similar responses. We report that a protein chimera (GFP170*) composed of the green fluorescent protein (GFP) fused to an internal fragment of the Golgi Complex Protein (GCP-170) forms nuclear aggregates analogous to those formed by polyQ proteins. Like the polyQ nuclear aggregates, GFP170* inclusions recruit molecular chaperones and proteasomal components, alter nuclear structures containing the promyelocytic leukemia protein (PML), recruit transcriptional factors such as CREB-binding protein (CBP) and p53, repress p53 transcriptional activity, and induce cell death. Our results indicate that nuclear aggregation and transcriptional effects are not unique to polyQ-containing proteins and may represent a general response to misfolded proteins in the nucleus.

Animals↗

PubMed2005

Golgi tethering factors.

Transport of cargo to, through and from the Golgi complex is mediated by vesicular carriers and transient tubular connections. In this review, we describe vesicle tethering events with the understanding that similar events occur during transport via larger structures. Tethering factors can be generally divided into a group of coiled-coil proteins and a group of multi-subunit complexes. Current evidence suggests that these factors function in a variety of membrane-membrane tethering events at the Golgi complex, interact with SNARE molecules, and are regulated by small GTPases of the Rab and Arl families.

Animals↗

PubMed2005

p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation.

Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.

3T3-L1 Cells↗

PubMed2005

Dissection of membrane dynamics of the ARF-guanine nucleotide exchange factor GBF1.

ADP-ribosylation factor (ARF)-facilitated recruitment of COP I to membranes is required for secretory traffic. The guanine nucleotide exchange factor GBF1 activates ARF and regulates ARF/COP I dynamics at the endoplasmic reticulum (ER)-Golgi interface. Like ARF and coatomer, GBF1 peripherally associates with membranes. ADP-ribosylation factor and coatomer have been shown to rapidly cycle between membranes and cytosol, but the membrane dynamics of GBF1 are unknown. Here, we used fluorescence recovery after photobleaching to characterize the behavior of GFP-tagged GBF1. We report that GBF1 rapidly cycles between membranes and the cytosol (t1/2 is approximately 17 +/- 1 seconds). GBF1 cycles faster than GFP-tagged ARF, suggesting that in each round of association/dissociation, GBF1 catalyzes a single event of ARF activation, and that the activated ARF remains on membrane after GBF1 dissociation. Using three different approaches [expression of an inactive (E794K) GBF1 mutant, expression of the ARF1 (T31N) mutant with decreased affinity for GTP and Brefeldin A treatment], we show that GBF1 is stabilized on membranes when in a complex with ARF-GDP. GBF1 dissociation from ARF and membranes is triggered by its catalytic activity, i.e. the displacement of GDP and the subsequent binding of GTP to ARF. Our findings imply that continuous cycles of recruitment and dissociation of GBF1 to membranes are required for sustained ARF activation and COP I recruitment that underlies ER-Golgi traffic.

ADP-Ribosylation Factors↗

PubMed2004

h-ERES-y in ER-Golgi transport.

A debate continues over whether the Golgi is a stable organelle or a transient manifestation of continuous membrane flow from specialized ER exit domains (ERES). A new study from daSilva and colleagues shows that in plant cells, individual Golgi always form adjacent to an existing ERES, and that an ERES and its associated Golgi stack move as a single secretory unit. Their results support a model in which Golgi biogenesis correlates with ERES function.

Animals↗

PubMed2003

Mutations in COCH that result in non-syndromic autosomal dominant deafness (DFNA9) affect matrix deposition of cochlin.

The COCH gene mutated in autosomal dominant sensorineural deafness (DFNA9) encodes cochlin, a major constituent of the inner ear extracellular matrix. Sequence analysis of cochlin from DFNA9 patients identified five distinct single-amino-acid mutations within a conserved region (the LCCL domain) of cochlin. To define the molecular basis of DFNA9, we have generated myc-tagged wild-type and mutant cochlins and explored their behavior in transient transfection systems. Western blotting of cell lysates and culture media indicates that wild-type and mutant cochlins are synthesized and secreted in similar amounts. Immunofluorescent staining confirms that all are detected within the endoplasmic reticulum and the Golgi complex of transfected cells. Our findings suggest that COCH mutations are unlikely to cause abnormalities in secretion and suggest that extracellular events might cause DFNA9 pathology. In agreement, we show that wild-type cochlin accumulates in extracellular deposits that closely parallel the matrix component fibronectin, whereas mutant cochlins vary in the amount and pattern of extracellular material. Whereas some mutants exhibit an almost normal deposition pattern, some show complete lack of deposition. Our results suggest that DFNA9 results from gene products that fail to integrate correctly into the extracellular matrix. The partial or complete penetrance of integration defects suggests that DFNA9 pathology may be caused by multiple molecular mechanisms, including compromised ability of cochlin to self-assemble or to form appropriate complexes with other matrix components.

Amino Acid Sequence↗

PubMed2003

ADP-ribosylation factor/COPI-dependent events at the endoplasmic reticulum-Golgi interface are regulated by the guanine nucleotide exchange factor GBF1.

ADP-ribosylation factor (ARF) mediated recruitment of COPI to membranes plays a central role in transport between the endoplasmic reticulum (ER) and the Golgi. The activation of ARFs is mediated by guanine nucleotide exchange factors (GEFs). Although several ARF-GEFs have been identified, the transport steps in which they function are still poorly understood. Here we report that GBF1, a member of the Sec7-domain family of GEFs, is responsible for the regulation of COPI-mediated events at the ER-Golgi interface. We show that GBF1 is essential for the formation, differentiation, and translocation of pre-Golgi intermediates and for the maintenance of Golgi integrity. We also show that the formation of transport-competent ER-to-Golgi intermediates proceeds in two stages: first, a COPI-independent event leads to the formation of an unstable compartment, which is rapidly reabsorbed in the absence of GBF1 activity. Second, the association of GBF1 with this compartment allows COPI recruitment and leads to its maturation into transport intermediates. The recruitment of GBF1 to this compartment is specifically inhibited by brefeldin A. Our findings imply that the continuous recruitment of GBF1 to spatially differentiated membrane domains is required for sustained membrane remodeling that underlies membrane traffic and Golgi biogenesis.

ADP-Ribosylation Factors↗

PubMed2003

COPI recruitment is modulated by a Rab1b-dependent mechanism.

The small GTPase Rab1b is essential for endoplasmic reticulum (ER) to Golgi transport, but its exact function remains unclear. We have examined the effects of wild-type and three mutant forms of Rab1b in vivo. We show that the inactive form of Rab1b (the N121I mutant with impaired guanine nucleotide binding) blocks forward transport of cargo and induces Golgi disruption. The phenotype is analogous to that induced by brefeldin A (BFA): it causes resident Golgi proteins to relocate to the ER and induces redistribution of ER-Golgi intermediate compartment proteins to punctate structures. The COPII exit machinery seems to be functional in cells expressing the N121I mutant, but COPI is compromised, as shown by the release of beta-COP into the cytosol. Our results suggest that Rab1b function influences COPI recruitment. In support of this, we show that the disruptive effects of N121I can be reversed by expressing known mediators of COPI recruitment, the GTPase ARF1 and its guanine nucleotide exchange factor GBF1. Further evidence is provided by the finding that cells expressing the active form of Rab1b (the Q67L mutant with impaired GTPase activity) are resistant to BFA. Our data suggest a novel role for Rab1b in ARF1- and GBF1-mediated COPI recruitment pathway.

ADP-Ribosylation Factor 1↗

PubMed2003

Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator.

Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.

COP-Coated Vesicles↗

PubMed2003

The membrane-tethering protein p115 interacts with GBF1, an ARF guanine-nucleotide-exchange factor.

The membrane-transport factor p115 interacts with diverse components of the membrane-transport machinery. It binds two Golgi matrix proteins, a Rab GTPase, and various members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Here, we describe a novel interaction between p115 and Golgi-specific brefeldin-A-resistant factor 1 (GBF1), a guanine-nucleotide exchange factor for ADP ribosylation factor (ARF). GBF1 was identified in a yeast two-hybrid screen, using full-length p115 as bait. The interaction was confirmed biochemically, using in vitro and in vivo assays. The interacting domains were mapped to the proline-rich region of GBF1 and the head region of p115. These proteins colocalize extensively in the Golgi and in peripheral vesicular tubular clusters. Mutagenesis analysis indicates that the interaction is not required for targeting GBF1 or p115 to membranes. Expression of the p115-binding (pro-rich) region of GBF1 leads to Golgi disruption, indicating that the interaction between p115 and GBF1 is functionally relevant.

Animals↗

PubMed2003

Membrane targeting of p115 phosphorylation mutants and their effects on Golgi integrity and secretory traffic.

The cytosolic phosphoprotein p115 is required for ER to Golgi traffic and for Golgi reassembly after mitosis. In cells, p115 is localized to ER exit sites, ER-Golgi Intermediate Compartment (ERGIC) and the Golgi, and cycles between these compartments. P115 is phosphorylated on serine 942, and this modification appears to control p115 association with membranes. P115 is likely to function by reversibly interacting with effector proteins, and in the Golgi, two proteins, GM130 and giantin, have been shown to bind p115. The GM130-p115 and the giantin-p115 interactions are enhanced by p115 phosphorylation. Phosphorylation appears to be essential for p115 function, since substitutions of serine 942 abolish p115 ability to sustain cisternal reformation in an in vitro assay reconstituting Golgi reassembly after mitosis. Here, we explored how phosphorylation of p115 affects its intracellular targeting to distinct cellular compartments, and its function in secretory traffic. We generated phosphorylation mutants of p115 and tested their ability to target to ER exit sites, ERGIC and the Golgi. In addition, we explored whether expression of the mutants causes disruption of Golgi structure and perturbs ER-Golgi traffic of a VSV-G cargo protein.

Animals↗

PubMed2002

Hassles with taking out the garbage: aggravating aggresomes.

Diverse human diseases ranging from amyloidosis to neurodegenerative diseases are now recognized as 'conformational diseases' caused by protein misfolding and protein aggregation. Misfolded and aggregated proteins are usually handled in the cell through chaperone-mediated refolding, or when that is impossible, destroyed by proteasomal degradation. Recent evidence suggests that cells might have evolved a third pathway that involves the sequestration of aggregated proteins into specialized 'holding stations' called aggresomes. The aggresomal pathway provides a mechanism by which aggregated proteins form particulate (approximately 200 nm) mini-aggregates that are transported on microtubules (MTs) towards the MT organizing center (MTOC) by a process mediated by the minus-end motor protein dynein. Once at the MTOC, the individual particles pack into a single, usually spherical aggresome (1-3 microm) that surrounds the MTOC. Aggresomes are dynamic: they recruit various chaperones and proteasomes, presumably to aid in the disposal of the aggregated proteins. In addition, the formation of an aggresome is likely to activate the autophagic clearance mechanism that terminates in lysosomal degradation. Hence, the aggresome pathway may provide a novel system to deliver aggregated proteins from the cytoplasm to lysosomes for degradation. Although it is clear that many pathological states correlate with the formation of aggresomes, their causal relationships remain hotly debated. Here, we describe the current state of our knowledge of the aggresome pathway and outline the open questions that provide the focus of current research.

Cytoskeleton↗

PubMed2002

A novel type of regulation of the vimentin intermediate filament cytoskeleton by a Golgi protein.

Whether the highly dynamic structure of the vimentin intermediate filament (IF) cytoskeleton responds to cues from cellular organelles, and what proteins might participate in such events is largely unknown. We have shown previously that the Golgi protein formiminotransferase cyclodeaminase (FTCD) binds to vimentin filaments in vivo and in vitro, and that overexpression of FTCD causes dramatic rearrangements of the vimentin IF cytoskeleton (Gao and Sztul, J. Cell Biol. 152, 877-894, 2001). Using real-time imaging, we now show that FTCD causes bundling of individual thinner vimentin filaments into fibers and that the bundling always originates at the Golgi. FTCD appears to be the molecular "glue" since FTCD cross-links vimentin filaments in vitro. To initiate the analysis of structural determinants required for FTCD function in vimentin dynamics, we used structure-based design to generate individual formiminotransferase (FT) and cyclodeaminase (CD) domains, and to produce an enzymatically inactive FTCD. We show that the intact octameric structure is required for FTCD binding to vimentin filaments and for promoting filament assembly, but that eliminating enzymatic activity does not affect FTCD effects on the vimentin cytoskeleton. Our findings indicate that the Golgi protein FTCD is a potent modulator of the vimentin IF cytoskeleton, and suggest that the Golgi might act as a reservoir for proteins that regulate cytoskeletal dynamics.

Ammonia-Lyases↗