Search

Membrane traffic between secretory and endosomal compartments is vesicle‐mediated and must be tightly balanced to maintain a physiological compartment size. Vesicle formation is initiated by guanine nucleotide exchange factors (GEFs) that activate the ARF family of small GTPases. Regulatory mechanisms, including reversible phosphorylation, allow ARF‐GEFs to support vesicle formation only at the right time and place in response to cellular needs. Here, we review current knowledge of how the Golgi‐specific brefeldin A‐resistance factor 1 (GBF1)/brefeldin A‐inhibited guanine nucleotide exchange protein (BIG) family of ARF‐GEFs is influenced by phosphorylation and use predictive paradigms to propose new regulatory paradigms. We describe a conserved cluster of phosphorylation sites within the N‐terminal domains of the GBF1/BIG ARF‐GEFs and suggest that these sites may respond to homeostatic signals related to cell growth and division. In the C‐terminal region, GBF1 shows phosphorylation sites clustered differently as compared with the similar configuration found in both BIG1 and BIG2. Despite this similarity, BIG1 and BIG2 phosphorylation patterns are divergent in other domains. The different clustering of phosphorylation sites suggests that the nonconserved sites may represent distinct regulatory nodes and specify the function of GBF1, BIG1, and BIG2. [ABSTRACT FROM AUTHOR]

Tethering factors regulate the targeting of membrane-enclosed vesicles under the control of Rab GTPases. p115, a golgin family tether, has been shown to participate in multiple stages of ER/Golgi transport. Despite extensive study, the mechanism of action of p115 is poorly understood. SNARE proteins make up the machinery for membrane fusion, and strong evidence shows that function of p115 is directly linked to its interaction with SNAREs. Using a gel filtration binding assay, we have demonstrated that in solution p115 stably interacts with ER/Golgi SNAREs rbet1 and sec22b, but not membrin and syntaxin 5. These binding preferences stemmed from selectivity of p115 for monomeric SNARE motifs as opposed to SNARE oligomers. Soluble monomeric rbet1 can compete off p115 from coat protein II ( COPII) vesicles. Furthermore, excess p115 inhibits p115 function in trafficking. We conclude that monomeric SNAREs are a major binding site for p115 on COPII vesicles, and that p115 dissociates from its SNARE partners upon SNAREpin assembly. Our results suggest a model in which p115 forms a mixed p115/ SNARE helix bundle with a monomeric SNARE, facilitates the binding activity and/or concentration of the SNARE at prefusion sites and is subsequently ejected as SNARE complex formation and fusion proceed. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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 fulllength 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]