Frank Adolf

Membrane curvature induced by Arf1-GTP is essential for vesicle formation.

The GTPase Arf1 is considered as a molecular switch that regulates binding and release of coat proteins that polymerize on membranes to form transport vesicles. Here, we show that Arf1-GTP induces positive membrane curvature and find that the small GTPase can dimerize dependent on GTP. Investigating a possible link between Arf dimerization and curvature formation, we isolated an Arf1 mutant that cannot dimerize. Although it was capable of exerting the classical role of Arf1 as a coat receptor, it could not mediate the formation of COPI vesicles from Golgi-membranes and was lethal when expressed in yeast. Strikingly, this mutant was not able to deform membranes, suggesting that GTP-induced dimerization of Arf1 is a critical step inducing membrane curvature during the formation of coated vesicles.

COPI Budding within the Golgi Stack

The Golgi serves as a hub for intracellular membrane traffic in the eukaryotic cell. Transport within the early secretory pathway, that is within the Golgi and from the Golgi to the endoplasmic reticulum, is mediated by COPI-coated vesicles. The COPI coat shares structural features with the clathrin coat, but differs in the mechanisms of cargo sorting and vesicle formation. The small GTPase Arf1 initiates coating on activation and recruits en bloc the stable heptameric protein complex coatomer that resembles the inner and the outer shells of clathrin-coated vesicles. Different binding sites exist in coatomer for membrane machinery and for the sorting of various classes of cargo proteins. During the budding of a COPI vesicle, lipids are sorted to give a liquid-disordered phase composition. For the release of a COPI-coated vesicle, coatomer and Arf cooperate to mediate membrane separation.

Coatomer and dimeric ADP ribosylation factor 1 promote distinct steps in membrane scission

During membrane budding, coatomer drives initial curvature of the bud, whereas dimeric Arf1 is necessary for membrane scission.

ArfGAP1 Activity and COPI Vesicle Biogenesis

Golgi‐derived coat protein I (COPI) vesicles mediate transport in the early secretory pathway. The minimal machinery required for COPI vesicle formation from Golgi membranes in vitro consists of (i) the hetero‐heptameric protein complex coatomer, (ii) the small guanosine triphosphatase ADP‐ribosylation factor 1 (Arf1) and (iii) transmembrane proteins that function as coat receptors, such as p24 proteins. Various and opposing reports exist on a role of ArfGAP1 in COPI vesicle biogenesis. In this study, we show that, in contrast to data in the literature, ArfGAP1 is not required for COPI vesicle formation. To investigate roles of ArfGAP1 in vesicle formation, we titrated the enzyme into a defined reconstitution assay to form and purify COPI vesicles. We find that catalytic amounts of Arf1GAP1 significantly reduce the yield of purified COPI vesicles and that Arf1 rather than ArfGAP1 constitutes a stoichiometric component of the COPI coat. Combining the controversial reports with the results presented in this study, we suggest a novel role for ArfGAP1 in membrane trafficking.

Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication

Spindle pole bodies (SPBs), like nuclear pore complexes, are embedded in the nuclear envelope (NE) at sites of fusion of the inner and outer nuclear membranes. A network of interacting proteins is required to insert a cytoplasmic SPB precursor into the NE. A central player of this network is Nbp1 that interacts with the conserved integral membrane protein Ndc1. Here, we establish that Nbp1 is a monotopic membrane protein that is essential for SPB insertion at the inner face of the NE. In vitro and in vivo studies identified an N‐terminal amphipathic α‐helix of Nbp1 as a membrane‐binding element, with crucial functions in SPB duplication. The karyopherin Kap123 binds to a nuclear localization sequence next to this amphipathic α‐helix and prevents unspecific tethering of Nbp1 to membranes. After transport into the nucleus, Nbp1 binds to the inner nuclear membrane. These data define the targeting pathway of a SPB component and suggest that the amphipathic α‐helix of Nbp1 is important for SPB insertion into the NE from within the nucleus.

Scission of COPI and COPII vesicles is independent of GTP hydrolysis.

Intracellular transport and maintenance of the endomembrane system in eukaryotes depends on formation and fusion of vesicular carriers. A seeming discrepancy exists in the literature about the basic mechanism in the scission of transport vesicles that depend on GTP‐binding proteins. Some reports describe that the scission of COP‐coated vesicles is dependent on GTP hydrolysis, whereas others found that GTP hydrolysis is not required. In order to investigate this pivotal mechanism in vesicle formation, we analyzed formation of COPI‐ and COPII‐coated vesicles utilizing semi‐intact cells. The small GTPases Sar1 and Arf1 together with their corresponding coat proteins, the Sec23/24 and Sec13/31 complexes for COPII and coatomer for COPI vesicles were required and sufficient to drive vesicle formation. Both types of vesicles were efficiently generated when GTP hydrolysis was blocked either by utilizing the poorly hydrolyzable GTP analogs GTPγS and GMP‐PNP, or with constitutively active mutants of the small GTPases. Thus, GTP hydrolysis is not required for the formation and release of COP vesicles.

Sec24C/D-isoform–specific sorting of the preassembled ER–Golgi Q-SNARE complex

SNAREs are incorporated into COPII vesicles by direct interaction with Sec24. In mammals, Sec24 isoforms recruit either Sec22b or the Q-SNARE complex comprising Syntaxin5, GS27, and Bet1. Analysis of immunoisolated COPII vesicles and intracellular localization of Sec24 isoforms indicates that all ER-Golgi SNAREs are present on the same vesicles.

Small G Proteins: Arf Family GTPases in Vesicular Transport

Small GTP-binding proteins of the ADP-ribosylation factor (Arf) family are key components of trafficking vesicles. In the past three decades a number of vesicular carriers, whose formation depends on members of the Arf family were identified, and general molecular mechanisms how these transport carriers form and operate were established. Here we describe discovery and roles of the Arf-dependent carriers of the early secretory pathway, the COPI and COPII vesicles. We will discuss their function with regard to molecular mechanisms in coat recruitment, selection of cargo proteins, vesicle membrane budding/scission, and vesicle uncoating.

Analysis of Golgi complex functions: in vitro reconstitution systems.

In vitro reconstitution is prerequisite to investigate complex cellular functions at the molecular level. Reconstitution systems range from combining complete cellular cytosol with organelle-enriched membrane fractions to liposomal systems where all components are chemically defined and can be chosen at will. Here, we describe the in vitro reconstitution of COPI-coated vesicles from semi-intact cells. Efficient vesicle formation is achieved by simple incubation of permeabilized cells with the minimal set of coat proteins Arf1 and coatomer, and guanosine trinucleotides. GTP hydrolysis or any mechanical manipulations are not required for efficient COPI vesicle release.

Core Proteome and Architecture of COPI Vesicles

Retrieval of escaped ER-residents and intra-Golgi transport is facilitated by coat protein complex I (COPI)-coated vesicles. Their formation requires the activated small GTPase ADP-ribosylation factor (Arf) and the coat complex coatomer. Here we assess the protein composition of COPI vesicles by combining stable isotope labeling with amino acids in cell culture (SILAC) with in vitro reconstitution of COPI vesicles from semi-intact cells (SIC) using the minimal set of recombinant coat proteins. This approach yields an unbiased picture of the proteome of these carriers. We define a set of ~40 proteins common to COPI vesicles produced from different human as well as murine cell lines. Almost all bona fide COPI vesicle proteins are either ER-Golgi cycling proteins or Golgi-residents, while only a minor portion of secreted proteins was found. Moreover, we have investigated a putative role of γ- and ζ-COP as well as Arf isoforms in sorting and recruitment of specific proteins into COPI vesicles. As opposed to the related COPII system, all isoforms of coatomer and all COPI-forming isoforms of the small GTPase Arf produce COPI-coated vesicles with strikingly similar protein compositions. We present a model for the core architecture of COPI vesicles.