Research Report 2000 - MDC

Functions of Dynamin
II and PKC in
Post-Golgi Vesicle
Formation
Peter Westermann
Cellular functions depend on proper
transport and correct subcellular
localization of proteins. To accomplish
this, secretory proteins, lysosomal
proteins and membrane proteins have
to be sorted in the trans-Golgi
network (TGN) and packed into
specific transport vesicles. Vesicle
formation is controlled by different G
protein families. Therefore, functions
of inhibitory trimeric G proteins and
of dynamin II have been studied. In
addition, the PKC-dependent
regulation of vesicle formation at the
TGN is being investigated by
analyzing Golgi-bound PKC
substrates.
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New methods for investigating
molecular interactions
K. Bulygin, A. Malygin, G. Karpova
(Novosibirsk), J. Dong
Two methods have been developed for
studying molecular interactions.
RNA-DNA interaction have been
studied by cross-linking (K. Bulygin
et al., 1998) while protein domain
interactions have been analyzed by
affinity binding of cytosolic or
membrane proteins to peptide-tagged
protein domains attached to agarose
matrices.
Interactions between dynamin II
domains and the Golgi
apparatus
J. Dong, M. Knoblich in
collaboration with A. Otto, E.-C.
Müller, and C. Lindschau (FVK)
The functions of dynamins depend on
their domain structure. To study the
binding of dynamin II to Golgi
membranes, the pleckstrin-homology
domain (PHD), the proline-rich
domain (PRD) and the C-terminal part
of dynamin II, consisting of PHD,
GTPase activator domain and PRD,
were expressed and purified.
Interactions between these domains
and cytosolic or membrane proteins
were studied by affinity binding and
cross-linking. PHD binds with high
affinity to Golgi membranes, but does
not interact with proteins suggesting
binding mainly to phospholipids. The
proteins that interact with PRD are
SH3 domain-containing proteins,
amphiphysin I , amphiphysin II and
SH3GL2, while additional proteins
may bind within larger complexes.
The nature and composition of these
protein complexes is presently being
studied. In addition, membranebound,
but not cytosolic profilin I
promotes attachment of dynamin II to
the Golgi apparatus and supports
transport vesicle formation (J. Dong et
al., in revision).
Identification of PKC substrates
attached to the Golgi apparatus
B. Radau, M. Knoblich in
collaboration with A. Otto,
E.-C. Müller
Stimulation of vesicular transport
between the TGN and plasma
membrane by activation of PKC
(Westermann et al., 1996) may depend
on the phosphorylation of Golgibound
proteins. MARCKS,
MacMARCKS, cytokeratin 8,
cytokeratin 18 and synaptobrevin 2
have been identified by in situ
phosphorylation, two-dimensional
protein electrophoresis and peptide
sequencing as Golgi-bound PKC
substrates. The impact of individual
phosphoproteins on vesicle formation
is under investigation.
Selected Publications
K. Bulygin, K., Malygin, A.,
Karpova, G., and Westermann, P.
(1998) Site-specific modification of
4.5S RNA apical domain by
complementary oligodeoxynucleotides
carrying an alkylating group. Eur. J.
Biochem. 251, 175 - 180.
Westermann, P., Knoblich, M., Maier,
O., Lindschau C., and Haller, H.
(1996) Protein kinase C bound to the
Golgi apparatus supports formation of
constitutive transport vesicles.
Biochem. J. 320, 561-568.
Structure of the Group
Group leader
Dr. Peter Westermann
Guest scientist
Dr. Olaf Maier
Graduate students
Jiaxin Dong
Boris Radau
Technical assistant
Maria Knoblich