Uwe Plessmann

Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins.

Mammalian neurofilament triplet proteins (68 K, 160 K and 200 K) have been correlated by a biochemical, immunological and protein chemical study. The 160 K and 200 K triplet proteins are intermediate filament proteins in their own right, since they reveal the alpha‐helical coiled‐coil rod domain analyzed in detail for the 68 K protein. Triplet proteins display two distinct arrays. Their amino‐terminal region built analogously to non‐neuronal intermediate filament proteins should allow a co‐polymerization process via the interaction of coiled‐coil domains. The extra mass of all triplet proteins is allocated to carboxy‐terminally located extensions of increasing size and unique amino acid sequences. These may provide highly charged scaffolds suitable for interactions with other neuronal components. Such a domain of 68 K reveals, in sequence analysis, 47 glutamic acids within 106 residues. The epitope recognized by a monoclonal antibody reacting probably with all intermediate filament proteins has been mapped. It is located within the last 20 residues of the rods, where six distinct intermediate filament proteins point to a consensus sequence.

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching.

Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

Maturation of nuclear lamin A involves a specific carboxy‐terminal trimming, which removes the polyisoprenylation site from the precursor; implications for the structure of the nuclear lamina

Lamin A, a nuclear lamina protein of differentiated cells, is synthesized as a precursor of the mature molecule. Protein sequencing of the carboxyterminal 14 kDa fragment shows a lack of the last 18 residues predicted by cDNA sequencing. The carboxy‐terminal proteolytic maturation explains previous biochemical results including the loss of the polyisoprenylation site now located to the CXXM motif at the end of the chain. This view and earlier results on lamin B predict multiple post‐translational modifications shared by lamins A and B. While retained by lamin B, which is present in all cells, they are lost by maturation from lamin A, which probably acts only as an additional lamina constituent in differentiated cells.

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled‐coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin‐like predecessor.

The amino acid sequence of protein II and its phosphorylation site for protein kinase C; the domain structure Ca2+ modulated lipid binding proteins

Protein II isolated from porcine intestinal epithelium is a Ca2+‐modulated lipid‐binding protein. The amino acid sequence of porcine protein II reported here sheds new light on the properties of a multigene protein family which includes the tyrosine kinase substrates of the sarc gene (p36) and of the EGF‐receptor (p35). The sequence consolidates the structural principle in which an amino‐terminal tailpiece of variable length is followed by a core built from four internally homologous segments for those proteins in the 35‐40 kd range. Sequence data also show that the core can now be described as two domains each containing one low and one high homology segment. This view accounts for two Ca2+ sites, lipid aggregation and F‐actin bundling–when present–and suggests that properties of the cores in which protein II differs from p36 and p35 arise primarily from segments 1 and 2. The protease‐sensitive tailpiece of protein II is very short and lacks the phosphorylatable tyrosine present in the larger tail domains of p36 and p35. It harbors, however, like the p36 domain, the major site for in vitro phosphorylation by the Ca2+‐ and lipid‐activated protein kinase C. In protein II this site is most likely threonine 6. The sequence alignment also explains why protein II does not interact with a unique p11, a property probably specific for p36. Our results further suggest that liver endonexin may reflect two protein species both closely related to protein II.

Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site.

Binding of AP180 to clathrin triskelia induces their assembly into 60‐70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N‐terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C‐terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1‐20). This protein, localized to synaptic termini, has so far been of unknown function.

Protein-chemical characterization of NF-H, the largest mammalian neurofilament component; intermediate filament-type sequences followed by a unique carboxy-terminal extension

NF‐H has the highest mol. wt. of the three mammalian neurofilament components (NF‐L, NF‐M, NF‐H). In spite of its unusually large mol. wt., estimated to be 200 K by gel electrophoresis, NF‐H contains sequences which identify it as an integral intermediate filament (IF) protein in its amino‐terminal region. We have isolated and partially characterized a basic, non‐α‐helical segment located at the amino‐terminal end with properties similar to headpieces of other non‐epithelial IF proteins. The highly α‐helical 40‐K fragment excised by chymotrypsin is now identified by the amino acid sequence of a 17‐K fragment. This sequence can be unambiguously aligned with the rod region of other IF proteins and covers about half of the presumptive coiled‐coil arrays. NF‐H and NF‐M show 45% sequence identity in this region. The extra mass of NF‐H in comparison with most other IF proteins arises from a carboxy‐terminal extension thought to be responsible for inter‐neurofilament cross‐bridges in axons. This autonomous domain has a unique amino acid composition characterized by a high content of proline, alanine and particularly of lysine and glutamic acid. The NF‐H tailpiece extension also carries a large number of serine phosphates, which are not evenly distributed, but are restricted to the amino‐terminal part. Having now delineated the intermediate filament‐type sequences for all three neurofilament proteins it seems very likely that the three components interact via coiled‐coil interactions. They all carry unique carboxy‐terminal extensions which increase in length from NF‐L to NF‐H and seem to extend from the filament wall.

Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension.

The sequence of the amino‐terminal 436 residues of porcine neurofilament component NF‐M (apparent mol. wt. in gel electrophoresis 160 kd), one of the two high mol. wt. components of mammalian neurofilaments, reveals the typical structural organization of an intermediate filament (IF) protein of the non‐epithelial type. A non‐alpha‐helical arginine‐rich headpiece with multiple beta‐turns (residues 1‐98) precedes a highly alpha‐helical rod domain able to form double‐stranded coiled‐coils (residues 99‐412) and a non‐alpha‐helical tailpiece array starting at residue 413. All extra mass of NF‐M forms, as a carboxy‐terminal tailpiece extension of approximately 500 residues, an autonomous domain of unique composition. Limited sequence data in the amino‐terminal region of this domain document a lysine‐ and particularly glutamic acid‐rich array somewhat reminiscent of the much shorter tailpiece extension of NF‐L (apparent mol. wt. 68 kd), the major neurofilament protein. NF‐M is therefore a true intermediate filament protein co‐polymerized with NF‐L via presumptive coiled‐coil type interactions and not a peripherally bound associated protein of a filament backbone built exclusively from NF‐L. Along the structurally conserved coiled‐coil domains the two neurofilament proteins show only approximately 65% sequence identity, a value similar to that seen when NF‐L and NF‐M are compared with mesenchymal vimentin. The highly charged and acidic tailpiece extensions of all triplet proteins particularly rich in glutamic acid seem unique to the neurofilament type of IFs. They could form extra‐filamentous scaffolds suitable for interactions with other neuronal components.(ABSTRACT TRUNCATED AT 250 WORDS)

The complete amino acid sequence of the major mammalian neurofilament protein (NF‐L)

The first complete amino acid sequence of a neurofilament protein has been established. Porcine NF‐L contains 548 residues corresponding to a molecular mass of ~62 kDa. This value is noticeably smaller than the 68–72 kDa estimates from gel electrophoresis. Sequence comparison among the 6 non‐epithelial intermediate filament (IF) proteins of warm‐blooded vertebrates shows that the three NF proteins are the most remote members. Additionally and unexpectedly they reveal among each other lower sequence identity than the three non‐neuronal IF proteins GFAP, desmin, and vimentin where the last two are particularly closely related. Certain schemes of IF protein evolution are discussed.

Posttranslational modifications of α- and β-tubulin in Giardia lamblia, an ancient eukaryote

Tubulin of Giardia lamblia, a representative of the oldest eukaryotes, was screened for posttranslational modifications. Mass spectrometry of the carboxy‐terminal peptides documents a large number of variants. Both α‐ and β‐tubulin show polyglycylation with up to 20 and 15 extra glycyl residues respectively. Minor variants show a low level of glutamylation without or with glycylation. The glutamylation‐specific antibody GT335 detects α‐ and β‐tubulin in immunoblots. The terminal tyrosine is fully retained in α‐tubulin, which is completely acetylated at Lys‐40. Thus except for the detyrosination/tyrosination cycle all posttranslational modifications known for higher eukaryotes are already present in Giardia.