Talk:2010 Lecture 5
Helicase activity is embedded within the ribosome
"Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization."
"This distinction between rough and smooth ER was made even more explicit by Palay and Palade (1955), who found that the so-called Nissl bodies in neurons were none other than clumps of rough ER, which were distinct but connected to sections of "a granular reticulum" or smooth ER." http://jcb.rupress.org/cgi/content/full/168/1/12-a?
single span membrane proteins
- Type II (cytoplasmic N-terminus/lumenal C-terminus)
LOCATE is a curated database that houses data describing the membrane organization and subcellular localization of proteins from the RIKEN FANTOM4 mouse and human protein sequence set. The membrane organization is predicted by the high-throughput, computational pipeline MemO. The subcellular locations were determined by a high-throughput, immunofluorescence-based assay and by manually reviewing peer-reviewed publications. http://locate.imb.uq.edu.au/
Prediction of non-classical protein secretion - The SecretomeP 2.0 server produces ab initio predictions of non-classical i.e. not signal peptide triggered protein secretion. The method queries a large number of other feature prediction servers to obtain information on various post-translational and localizational aspects of the protein, which are integrated into the final secretion prediction. http://www.cbs.dtu.dk/services/SecretomeP/
secretory signal peptide (SP)
- targets a protein for translocation across the plasma membrane in prokaryotes and across the endoplasmic reticulum (ER) membrane in eukaryotes
- N-terminal peptide 15–30 amino acids long cleaved off during translocation of the protein across the membrane
- no simple consensus sequence
The Golgi predictor is implemented using Type II transmembrane proteins only. Its application to Type I, multi-spanning or N/C-terminal anchored membrane proteins is yet to be validated. http://ccb.imb.uq.edu.au/golgi/golgi_predictor.shtml Yuan Z, Teasdale RD. Prediction of Golgi Type II membrane proteins based on their transmembrane domains. Bioinformatics. 2002 Aug; 18(8): 1109-15.
peroxisomal targeting sequence (PTS) at the extreme COOH terminus of human catalase. The last four amino acids of this protein (-KANL) are necessary and sufficient to effect targeting to peroxisomes in both human fibroblasts and Saccharomyces cerevisiae, when appended to the COOH terminus of the reporter protein, chloramphenicol acetyl transferase. PMID: 8769411
Differential use of signal peptides and membrane domains is a common occurrence in the protein output of transcriptional units. Davis MJ, Hanson KA, Clark F, Fink JL, Zhang F, Kasukawa T, Kai C, Kawai J, Carninci P, Hayashizaki Y, Teasdale RD. PLoS Genet. 2006 Apr;2(4):e46. Epub 2006 Apr 28. PMID: 16683029
- "Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi-spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units (TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our high-confidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome."
Nuclear Localization Sequence (NLS)
two clusters of basic amino acids, separated by a spacer of about 10 amino acids
- signals are recognized by importin α. Importin α contains a bipartite NLS itself, which is specifically recognized by importin β
Demonstration that the nuclear-localization signal (NLS) of the SV40 large T-antigen can direct a cytoplasmic protein to the cell nucleus http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A2922&rendertype=figure&id=A2934
Nuclear Export Sequence (NES)
- short sequence of 5-6 hydrophobic amino acids
- Myosin V may help partition the ER Wöllert et al. find that, during mitosis, myosin V-driven movement of small globular vesicles along F-actin is strongly inhibited, but the movement of ER and ER network formation on F-actin is up-regulated. Thus F-actin may help partition the ER during cell division.
- Peroxisomes segregate using Myo2p Hoepfner et al. analyzed the movement of peroxisomes in budding yeast. In the absence of the dynamin-like protein Vps1p only one or two giant peroxisomes remained, but segregation still occurred. Peroxisome movement was abolished by latrunculin A treatment, and movement was also found to be dependent on the myosin motor Myo2p.
- Golgi clusters partition during mitosis Golgi partitioning during mitosis has been suggested to occur via fusion of the Golgi with the endoplasmic reticulum. In contrast, Jokitalo et al. find that the Golgi membranes split into small clusters that persist through mitosis and partition early in mitosis to two sides of the nucleus. These two sets of clusters are pushed apart during anaphase, and form the basis for the re-formation of the Golgi in the two daughter cells.
- Visualizing the location and dynamics of exocytosis Schmoranzer et al. use total internal reflection (TIR) fluorescence microscopy to visualize exocytosis in mammalian cells (e.g., see event on left side of video). The analysis reveals that there are no preferred sites for constitutive exocytosis in this system.
- Visualizing the location and dynamics of exocytosis Toomre et al. use a combination of TIR microscopy (green, labeling molecules close to or at the membrane) and standard fluorescence microscopy (red, for molecules further from the membrane) to visualize [/content/vol149/issue1/images/data/33/DC1/Fig_1b.mov trafficking to and fusion with] the plasma membrane during exocytosis. Red dots turn yellow then green as they approach the membrane, and then explode in a burst of light as they fuse with the plasma membrane during exocytosis. The transport containers appear to be partially anchored at the membrane before fusion, and can undergo either partial or complete fusion events.
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