Identification and localization of a beta-COP-like protein involved in the morphodynamics of the plant Golgi apparatus.

This paper examines the molecular machinery involved in membrane exchange within the plant endomembrane system. A study has been undertaken on beta-COP-like proteins in plant cells using M3A5, an antibody raised against the conserved sequence of mammalian beta-COP proteins. In mammalian cells, beta-COP proteins are part of a complex named the coatomer, which probably recruits some specific areas of the endomembrane system. Immunofluorescence analyses by confocal laser scanning microscopy showed that beta-COP-like proteins marked predominantly the plant Golgi apparatus. Other proteins known to be part of a potential machinery for COPI vesicle formation (gamma-COP, beta'-COP and Arf1 proteins) were immunolocalized on the same membraneous structures as beta-COP. Moreover, beta-COP and other COPI antibodies stained the cell plate in dividing cells. It is further shown that, in maize root cells, and in contrast to observations upon mammalian cells, the drug Brefeldin A (BFA) does not induce the release of beta-COP and Arf1 proteins from the Golgi membrane into the cytosol. These data clearly demonstrate that the antibody M3A5 is a valuable marker for studies on trafficking events in plant cells. They also report for the first time the location of COP components in plant tissue at the light level, especially on a model well known for secretion, i.e. the maize root cells. They also suggest that the membrane recruitment machinery may function in a plant-specific way.

Screen and identification of proteins interacting with ADAM19 cytoplasmic tail.

ADAM family plays important roles in neurogenesis. The cytoplasmic tail of ADAM19 (ADAM19-CT) contains 193 residues. The presence of two putative SH3 ligand-binding sites suggests potential interactions with cytosolic proteins, which could be possibly linked to the functions of ADAM19. To address these issues, a yeast two-hybrid screen was performed in human fetal brain cDNA library to isolate proteins that interact with the cytoplasmic tail of ADAM19. Four proteins were obtained, ArgBP1, beta-cop, ubiquitin and a novel protein. GST-Pulldown assay has confirmed the interaction between AdAM19 and ArgBP1. By constructing series of deletion mutants of ADAM19-CT and ArgBP1 respectively, the interaction regions have been identified. They are the SH3 binding sites in ADAM19-CT and the P4 region in ArgBP1. And the interaction is specific. ArgBP1 does not bind to ADAM22, ADAM29 or ADAM9 (mouse). ArgBP1 may be the key protein, which accounts for the physiological function of ADAM19.

Phylogenetic relationships of the seven coat protein subunits of the coatomer complex, and comparative sequence analysis of murine xenin and proxenin.

The coatomer complex is involved in intracellular protein transport and comprises an assembly of seven polypeptide subunits designated alpha, beta, beta', gamma, delta, epsilon, and zeta COP. Rooted phylogenetic trees constructed from the full-length cDNA and amino acid sequences of 49 COP entities in different eukaryotes from yeast to man generally revealed striking conservation of each subunit through evolution. Both nucleotide and protein trees displayed close relationships between alpha and beta' subunits, between beta and gamma subunits, and between delta and zeta subunits, implying evolution from common ancestors as well as functional similarity. Interestingly, although 6 out of 7 epsilon-COP genes appeared to be grouped and related to the beta-COP genes, 4 out of 7 epsilon

The coatomer of Trypanosoma brucei.

Coatomer is a multisubunit complex involved in trafficking of vesicles between the endoplasmatic reticulum and the Golgi apparatus. From sequence homologies, all seven subunits, alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP, are encoded in the genome of Trypanosoma brucei. The complete predicted amino-acid sequences of beta-, beta'-, and zeta-COP show only 20-30% identity with higher eucaryotic homologues. The trypanosome coatomer complex was partially purified using a procedure similar to that used for bovine coatomer.

Glyceraldehyde-3-phosphate dehydrogenase is required for vesicular transport in the early secretory pathway.

Protein transport in the early secretory pathway requires Rab2 GTPase. This protein promotes the recruitment of soluble components that participate in protein sorting and recycling from pre-Golgi intermediates (vesicular tubular clusters (VTCs)). We previously reported that a constitutively activated form of Rab2 (Q65L) as well as Rab2 wild type promoted vesicle formation from VTCs. These vesicles contained Rab2, beta-COP, p53/gp58, and protein kinase Ciota/lambda but lacked anterograde-directed cargo. To identify other candidate Rab2 effectors, the polypeptide composition of the vesicles was further analyzed. We found that vesicles released in response to Rab2 also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To study the relationship of this enzyme to Rab2 function, we performed a quantitative binding assay to measure recruitment of GAPDH to membrane when incubated with Rab2. Rab2-treated microsomes showed a 5-10-fold increase in the level of membrane-associated GAPDH. We generated an affinity-purified anti-GAPDH polyclonal to study the biochemical role of GAPDH in the early secretory pathway. The antibody arrests transport of a reporter molecule in an assay that reconstitutes ER to Golgi traffic. Furthermore, the affinity-purified antibody blocked the ability of Rab2 to recruit GAPDH to membrane. However, the antibody did not interfere with Rab2 stimulated vesicle release. These data suggest that GAPDH is required for ER to Golgi transport. We propose that membranes incubated with anti-GAPDH and Rab2 form "dead end" vesicles that are unable to transport and fuse with the acceptor compartment.