concluded that the capacity of the proteasomal protein degradation system is by far not
fully used in the absence of proteotoxic stress.
Another cytoplasmic macromolecular assembly that proved highly suitable for
structural analysis using CET and subtomogram analysis is the axoneme cytoskeletal
element in eukaryotic cilia and flagella. An axoneme typically consists of a ring of
nine outer microtubule doublets with each an outer and inner dynein arm, connected
to two central microtubule doublets via nine radial spokes. Subtomogram averages
revealed nucleotide-induced global structural changes of the outer and inner dynein
arms, which drive sliding motions of adjacent microtubules and explain axoneme
bending [35, 36]. Further studies aimed at a detailed structural dissection of the
molecular axoneme architecture, focusing on the radial spokes [37], the
nexin-dynein regulatory complex that bridges outer microtubule doublets [38], the
polarity and asymmetry in outer dynein arms [39, 40] and structural defects of
axonemes arising from primary ciliary dyskinesia [41].
9.7.3 Membrane Protein Complexes
CET and subtomogram analysis are particularly attractive for studying the structure
of membrane-embedded and –associated complexes, because detergent solubilization is not required. Thus, destabilization of the complex as well as possible misinterpretation of the density of the detergent micelle surrounding a solubilized
membrane protein are avoided. Structural analysis of the nuclear pore complex
(NPC), the gateway for nucleo-cytoplasmic transport, has long remained an enormous challenge. The NPC is a highly dynamic, gigantic machinery of >100 MDa
molecular weight, embedded into the two membranes constituting the nuclear
envelope. CET and subtomogram analysis revealed that the *30 different nucleoporins, constituting the NPC, assemble into distinct cytoplasmic and nuclear rings,
associated to a scaffold [42, 43]. NPC structures from different organisms illuminated various aspects in regulation/gating of nucleo-cytoplasmic transport and led
to increasingly detailed insights into the molecular architecture of the NPC [44–47].
Protein transport between organelles is mediated by trafficking vesicles that are
formed by protein coats polymerizing as cages on the membrane surface. The
variable size of trafficking vesicles and the highly dynamic nature of coat proteins
precluded their analysis using conventional structural biology methods. Retrograde
transport from the Golgi to the ER and within the Golgi compartments is mediated
by coat protein complex (COP) I. Studies using CET and subtomogram analysis
revealed a three-fold symmetrical arrangement of the COPI components on the
vesicle membrane. COPI triads adopted alternative conformations to change the
number of neighboring interacting triads to form vesicles with variable size [48].
Based on a higher resolution subtomogram average, the detailed molecular architecture of a single COPI triad could be determined [49]. Anterograde transport of
newly synthesized proteins from the ER to the Golgi is mediated by COPII.
A recent study using subtomogram analysis revealed, how the inner layer of the
250
S. Pfeffer and F. Förster
fully used in the absence of proteotoxic stress.
Another cytoplasmic macromolecular assembly that proved highly suitable for
structural analysis using CET and subtomogram analysis is the axoneme cytoskeletal
element in eukaryotic cilia and flagella. An axoneme typically consists of a ring of
nine outer microtubule doublets with each an outer and inner dynein arm, connected
to two central microtubule doublets via nine radial spokes. Subtomogram averages
revealed nucleotide-induced global structural changes of the outer and inner dynein
arms, which drive sliding motions of adjacent microtubules and explain axoneme
bending [35, 36]. Further studies aimed at a detailed structural dissection of the
molecular axoneme architecture, focusing on the radial spokes [37], the
nexin-dynein regulatory complex that bridges outer microtubule doublets [38], the
polarity and asymmetry in outer dynein arms [39, 40] and structural defects of
axonemes arising from primary ciliary dyskinesia [41].
9.7.3 Membrane Protein Complexes
CET and subtomogram analysis are particularly attractive for studying the structure
of membrane-embedded and –associated complexes, because detergent solubilization is not required. Thus, destabilization of the complex as well as possible misinterpretation of the density of the detergent micelle surrounding a solubilized
membrane protein are avoided. Structural analysis of the nuclear pore complex
(NPC), the gateway for nucleo-cytoplasmic transport, has long remained an enormous challenge. The NPC is a highly dynamic, gigantic machinery of >100 MDa
molecular weight, embedded into the two membranes constituting the nuclear
envelope. CET and subtomogram analysis revealed that the *30 different nucleoporins, constituting the NPC, assemble into distinct cytoplasmic and nuclear rings,
associated to a scaffold [42, 43]. NPC structures from different organisms illuminated various aspects in regulation/gating of nucleo-cytoplasmic transport and led
to increasingly detailed insights into the molecular architecture of the NPC [44–47].
Protein transport between organelles is mediated by trafficking vesicles that are
formed by protein coats polymerizing as cages on the membrane surface. The
variable size of trafficking vesicles and the highly dynamic nature of coat proteins
precluded their analysis using conventional structural biology methods. Retrograde
transport from the Golgi to the ER and within the Golgi compartments is mediated
by coat protein complex (COP) I. Studies using CET and subtomogram analysis
revealed a three-fold symmetrical arrangement of the COPI components on the
vesicle membrane. COPI triads adopted alternative conformations to change the
number of neighboring interacting triads to form vesicles with variable size [48].
Based on a higher resolution subtomogram average, the detailed molecular architecture of a single COPI triad could be determined [49]. Anterograde transport of
newly synthesized proteins from the ER to the Golgi is mediated by COPII.
A recent study using subtomogram analysis revealed, how the inner layer of the
250
S. Pfeffer and F. Förster