Elizabeth Sztul

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 (∼ 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 μm) 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.

Listeria monocytogenes ActA-mediated escape from autophagic recognition

Autophagy degrades unnecessary organelles and misfolded protein aggregates, as well as cytoplasm-invading bacteria. Nevertheless, the bacteria Listeria monocytogenes efficiently escapes autophagy. We show here that recruitment of the Arp2/3 complex and Ena/VASP, via the bacterial ActA protein, to the bacterial surface disguises the bacteria from autophagic recognition, an activity that is independent of the ability to mediate bacterial motility. L. monocytogenes expressing ActA mutants that lack the ability to recruit the host proteins initially underwent ubiquitylation, followed by recruitment of p62 (also known as SQSTM1) and LC3, before finally undergoing autophagy. The ability of ActA to mediate protection from ubiquitylation was further demonstrated by generating aggregate-prone GFP–ActA–Q79C and GFP–ActA–170* chimaeras, consisting of GFP (green fluorescent protein), the ActA protein and segments of polyQ or Golgi membrane protein GCP170 (ref. 6). GFP–ActA–Q79C and GFP–ActA–170* formed aggregates in the host cell cytoplasm, however, these ActA-containing aggregates were not targeted for association with ubiquitin and p62. Our findings indicate that ActA-mediated host protein recruitment is a unique bacterial disguise tactic to escape from autophagy.

Accumulation of Virion Tegument and Envelope Proteins in a Stable Cytoplasmic Compartment during Human Cytomegalovirus Replication: Characterization of a Potential Site of Virus Assembly

ABSTRACT The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with thetrans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.

Human Cytomegalovirus pp28 (UL99) Localizes to a Cytoplasmic Compartment Which Overlaps the Endoplasmic Reticulum-Golgi-Intermediate Compartment

ABSTRACT Although the assembly of herpesviruses has remained an active area of investigation, considerable controversy continues to surround the cellular location of tegument and envelope acquisition. This controversy is particularly evident when the proposed pathways for α- and β-herpesvirus assembly are compared. We have approached this aspect of human cytomegalovirus (HCMV) assembly, specifically, envelopment, by investigating the intracellular trafficking of viral tegument proteins which localize in the cytoplasms of infected cells. In this study we have demonstrated that the virion tegument protein pp28 (UL99), a true late protein, was membrane associated as a result of myristoylation. A mutation in this protein which prevented incorporation of [3H]myristic acid also altered the detergent solubility and intracellular distribution of the protein when it was expressed in transfected cells. Using a panel of markers for intracellular compartments, we could localize the expression of wild-type pp28 to an intracellular compartment which colocalized with the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC), a dynamic compartment of the secretory pathway which interfaces with both the ER and Golgi apparatus. The localization of this viral tegument protein within an early secretory compartment of the cell provided further evidence that the assembly of the HCMV tegument likely includes a cytoplasmic phase. Because pp28 has been shown to be localized to a cytoplasmic assembly compartment in HCMV-infected cells, our findings also suggested that viral tegument protein interactions within the secretory pathway may have an important role in the assembly of the virion.

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.

Dissecting the role of the ARF guanine nucleotide exchange factor GBF1 in Golgi biogenesis and protein trafficking

COPI recruitment to membranes appears to be essential for the biogenesis of the Golgi and for secretory trafficking. Preventing COPI recruitment by expressing inactive forms of the ADP-ribosylation factor (ARF) or the ARF-activating guanine nucleotide exchange factor GBF1, or by treating cells with brefeldin A (BFA), causes the collapse of the Golgi into the endoplasmic reticulum (ER) and arrests trafficking of soluble and transmembrane proteins at the ER. Here, we assess COPI function in Golgi biogenesis and protein trafficking by preventing COPI recruitment to membranes by removing GBF1. We report that siRNA-mediated depletion of GBF1 causes COPI dispersal but does not lead to collapse of the Golgi. Instead, it causes extensive tubulation of the cis-Golgi. The Golgi-derived tubules target to peripheral ER-Golgi intermediate compartment (ERGIC) sites and create dynamic continuities between the ERGIC and the cis-Golgi compartment. COPI dispersal in GBF1-depleted cells causes dramatic inhibition of the trafficking of transmembrane proteins. Unexpectedly, soluble proteins continue to be secreted from GBF1-depleted cells. Our findings suggest that a secretory pathway capable of trafficking soluble proteins can be maintained in cells in which COPI recruitment is compromised by GBF1 depletion. However, the trafficking of transmembrane proteins through the existing pathway requires GBF1-mediated ARF activation and COPI recruitment.

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.

Role of vesicle tethering factors in the ER–Golgi membrane traffic

Tethers are a diverse group of loosely related proteins and protein complexes grouped into three families based on structural and functional similarities. A well‐accepted role for tethering factors is the initial attachment of transport carriers to acceptor membranes prior to fusion. However, accumulating evidence indicates that tethers are more than static bridges. Tethers have been shown to interact with components of the fusion machinery and with components involved in vesicle formation. Tethers belonging to the three families act at the same stage of traffic, suggesting that they mediate distinct events during vesicle tethering. Thus, multiple tether‐facilitated events are required to provide selectivity to vesicle fusion. In this review, we highlight findings that support this model.

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.

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 sec12 ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13 ts strain (mutant in the Sec13p component of COPII), and sec23 ts 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.