Protein import, nucleus

Heme (C34H3204N4Fe) represents an iron-porphyrin complex that has a protoporphyrin nucleus. Many important proteins contain heme as a prosthetic group. Hemoglobin is the quantitatively most important hemoprotein. Others are cytochromes (present in the mitochondria and the endoplasmic reticulum), catalase and peroxidase (that react with hydrogen peroxide), soluble guanylyl cyclase (that converts guanosine triphosphate, GTP, to the signaling molecule 3, 5 -cyclic GMP) and NO synthases. 

Hicks, G., and Raikhel, N. (1995). Protein import into the nucleus an integrated view. Annu. Rev. Cell Dev. Biol. 11, 55-88. 

Fig. 2. Macroscopic and microscopic structure of muscle (a) Entire muscle and its cross-section with fatty septa, (b) Fascicle with several muscle fibres (cells). A layer of fat along the fascicle is indicated, (c) Striated myofibre corresponding with one single muscle cell containing several nuclei. The lengths of a myofibre can be several tens of centimetres, (d) Myofibril inside a myocyte. It is one contractile element and contains actin and myosin and further proteins important for the muscular function, (e) Electron myograph of human skeletal muscle showing the band structure caused by the contractile myofilaments in the sarcomeres. One nucleus (Nu) and small glycogen granules (arrow, size <0.1 pm) are indicated.

Dendritic derivatives of these macrocycles can be placed in the wider context of studies on metalloporphyrins with sterically hindered faces which have been designed in attempts to mimic the properties of heme proteins and chlorophylls, and there are suggestions that steric isolation of the metalloporphyrin nucleus is important in certain biological functions, The redox properties of metalloporphyrins are well-documented they are dominated by two, reversible one-electron transfers involving both the metal and the ligand. The first dendritic porphyrins of general structure 47 and their Zn complexes were reported by Inoue et al. who... 

The final principal component of the cell is the nucleus. This is located in the center of the cell and is surrounded by a double membrane, the outer layer being derived from the ER of the cytoplasm and the inner layer coming from the nucleus itself. The two leaflets of the double membrane are fused in places, producing nuclear pores that enable the transfer of macromolecules from the cytoplasm to the nucleus. Two important components of the nucleus are chromatin and the nucleolus. Chromatin represents polymers of DNA complexed with protein. The nucleolus is a complex substructure, composed of ribonucleoprotein granules, that controls the synthesis of RNA destined to form the ribosomes of the cytoplasm. Cells engaged heavily in protein synthesis have... 

In comparison to signaling pathways which utilize transmembrane receptors (see chapter 5, 8,11), signahng via nuclear receptors is of relatively simple structure. The pathways lead directly, with only a few participating protein components, from the extracellular space to the level of the DNA in the nucleus. Most important protein components of the signal pathway are known and well characterized. Nevertheless, we understand very little of the mechanism by which the activated receptors lead to a transcription initiation. This is due to the extreme complexity of transcription initiation in eucaryotes (see 1.2). Both the variety of proteins involved in the formation of a competent initiation complex, as well as the influence of chromatin structure, make it difficult to elucidate the exact function of nuclear receptors in transcription initiation. 

Figure 12.8 Model for general and cell-specific plasmid nuclear import. (A) SV40 enhancer-mediated nuclear import. Because the transcription factors bound by this DNA sequence are ubiquitously expressed, SV40 DNA localizes to the nuclei of all cell types (see Table 12.1). (B) Smooth muscle-specific plasmid nuclear import. Smooth muscle-specific transcription factors, including SRF among others, can bind to their target sites within the SMGA promoter carried on a plasmid and serve to transport the DNA to the nucleus via interactions with the NLS-mediated protein import machinery. Since these factors are not expressed in other cell types, no nuclear import will occur in non-smooth muscle cells.

This view amounts to mitochondrial genes being stuck where they are because of an insuperable difficulty if translocating hydrophobic proteins between subcelluar compartments. Yet there seems to be no evidence that hydrophobicity presents a particular barrier to protein import. For example, mitochondrial ADP-ATP carriers (AACs) are intrinsic to the mitochondrial inner membrane, have six transmembrane helices, and yet are encoded in the nucleus (Saraste and Walker 1982 van der Giezen et al. 2002). 

Moore MS, BLOBH, G. The GTP binding protein ran/TC4 is required foe protein import into the nucleus. Nature36S 661-663,1993. 

An intensely investigated trophic factor is IGF-I, a pleiotropic protein important for normal development and maintenance of central and peripheral nervous system tissues. It interacts with the tyrosine kinase IGF-IR consisting of two extracellular a-subunits linked via disulfide bonds and two P-subunits, which are largely cytoplasmic and contain the tyrosine kinase domains. When activated by its substrate, IGF-I the IGF-IR signals to its major substrates, i.e., insulin receptor substrate-1 and -2 (IRS-1, IRS-2) and to She and from there on via the Ras/Raf pathway, via MEK and MAPK to the nucleus (Rubin and Baserga,... 

Proteins Imported to or exported from the nucleus contain a specific amino acid sequence that functions as a nuclear-localization signal (NLS) or a nuclear-export signal (NFS). Nucleus-restricted proteins contain an NLS but not an NES, whereas proteins that shuttle between the nucleus and cytoplasm contain both signals. 

Fig. 2 Uptake of nuclear proteins by an in virro-assembled nucleus. Permeabilized Xenopus sperm were incubated with an egg extract containing a GFP-NLS fusion protein for 30 min. Samples were stained with DAPl (A) or examined for GFP fluorescence (B). The fully assembled nucleus can import GFP-NLS whereas an assembly intermediate cannot. Scale bar, 10 pm.

Ever since the demonstration that synthetic peptides could target a protein to the nucleus when cross-linked to it (Goldfarb et ai, 1986), peptide-protein conjugates have been used widely in nuclear protein import assays in vivo and in vitro. The method described here is for the preparation of a fluorescent bovine serum albumin, SV40 NLS conjugate. However, this basic protocol can be adapted easily to allow the conjugation to other proteins, such as the naturally fluorescent protein allophycocyanin (Adam etai, 1991). 

Moore, M. S., and Blobel, G. (1993). The CTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature (London) 365, 661-663. 

Palacios, I., Weis, K Klebe, C., Mattaj, I. W., and Dingwall, C. (19%). RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus. J. Cell Biol. 133,485-494. 

It was demonstrated also that the acidic proteins stimulate synthesis in isolated chromatin fractions and increase the transcriptional activity of the DNA-histone complex (Frenster, 1965 Teng et al., 1970). These proteins promote the transcription of free DNA (Allfrey et al., 1972), and they determine transcriptional specificity. The accumulation of acidic nucleus proteins with a molecular weight of20,000-40,000 occurs during puff formation in Drosophila salivary gland chromosomes (Berendes, 1972). An important role in this process is played by phosphoproteins which demonstrate a high level of tissue specificity (Allfrey et al., 1972 Rickwood et al., 1972 Fig. 60). 

---