Abstract
To study the impact of dynamin II, a 100 kD GTP-binding protein, and of its individual domains on vesicle
biogenesis at the TGN, the pleckstrin-homology domain (PHD), the proline-rich domain (PRD) and the C-terminal
part of dynamin II (PCP)-comprising PHD, a coiled-coil domain and PRD-were expressed in E.coli (Dong et al.,
2000b). Analysis of fluorescence and CD spectra indicates that PHD is a compact globular domain with 20%
a-helix and 52% b-structure. The PHD binds to membrane lipids as well as to plasma membrane and Golgi
preparations, which suggests that PHD may have a major impact on membrane binding of dynamin II.
CD and
fluorescence spectra of the PRD indicate a low content of secondary structure and the PRD was therefore classified
as an unfolded domain. The PRD interacted weakly with lipids and membranes and binding efficiency was
stimulated by addition of cytosolic proteins. Specific binding of PRD may depend on the interaction with proteins
containing SH3 domains, like amphiphysin I and II as well as syndapin II, which were identified as major binding
proteins. In addition, binding of dynamin II to Golgi membranes depends on profilin I (Dong et al., 2000a).
To
investigate the effects of PHD and PRD on vesicle biogenesis, purified domains were added to in vitro budding
assays and the formation of constitutive transport vesicles at the TGN was measured. While the PHD was neither
inhibitory nor stimulatory, addition of PRD inhibited vesicle formation by 45%. The inhibition was reversed by
addition of the SH3 domain of amphiphysin II. The essential role of the PRD in the dynamin II-mediated vesicle
formation is underlined by the finding that an antibody recognizing the C-terminus of dynamin II was also strongly
inhibitory. To support these results in an in vivo system, the secretion of pulse-labeled heparansulfate proteoglycans
by 293 cells which overexpress PHD, PRD or PCP under control of the tet repressor was measured: in PHD
expressing cells, the early secretion was reduced by 27%, in PRD-expressing cells by 39% and in PCP-expressing
cells by 58%. The results indicate that both PRD and PHD are required for proper function of dynamin II in vivo.
To elucidate the mechanism(s) by which the PRD supports vesicle biogenesis, proteins which bind to
recombinant PRD were identified: amphiphysin I and II as well as syndapin II directly interacted with PRD via their
SH3 domains and might mediate indirect binding of adaptor protein complexes AP-1 (identified by g-adaptin), AP-2
(by a-adaptin) and AP-3 (by p47A). In addition, the endosome-associated protein EEA1 and b-tubulin were
identified as binding proteins of PRD.
Binding of three adaptor protein complexes provides evidence that
dynamin II is involved in exocytic and endocytic pathways. Accordingly, dynamin II was localized in HeLa cells to
the Golgi apparatus, to the plasma membrane and to spotted cytoplasmic structures, evidently vesicles. The extent of
colocalization between dynamin II and g-adaptin, a-adaptin or p47A differs at individual sites. PHD and PRD may
have distinct effects on the distribution of dynamin II within the cell: the EGFP-PHD fusion protein localized to the
Golgi area as well as to the plasma membrane of HeLa cells, whereas EGFP-PRD was predominantly localized to
the perinuclear area including the Golgi apparatus.
The inhibition of vesicle formation in vitro by PRD and the
inhibition of the secretion in vivo by expressed PRD or PCP may originate from a competition between the PRD
domain and dynamin II for binding proteins, which was shown to result in a detachment of dynamin II from the
Golgi. As a second effect, an inhibition of (in vitro) dynamin II oligomer assembly was noted when PRD or PCP
were overexpressed. The requirement of the coiled-coil domain for self-assembly, published by Sever et al. (Sever
et al., 1999), is supported by the observation that EGFP-PCP but not EGFP-PRD accumulated in large vacuoles in
293 cells, which suggests that PCP forms stable oligomers and attaches predominantly to a membrane-coated
compartment.
The combination of data obtained by different methods, e.g., in vitro vesicle formation, protein
affinity binding and studies on intracellular localizations have provided compelling evidence that dynamin II-in
addition to its known function in endocytosis-is also required for exocytic and intracellular vesicle transport
pathways. The functions of dynamin II during vesicle formation at the TGN depend on interactions with membranes
via the PHD, on binding of SH3 domain proteins or polyproline-binding proteins by the PRD and on oligomerization
of dynamin II for which the PRD and the coiled-coil domain are required. |
