No abstract is provided for this article.
This chapter reviews the current knowledge of membrane proteins and explains that they are handled by different targeting-translocation machineries from the perspective of possible structural constraints imposed by these machineries. This point of view has not been much elaborated up to now and there are not many clear examples of structural constraints that go beyond those imposed by the lipid bilayer itself. It also argues that such constraints do exist and that they cannot be ignored if one wishes to fully understand the principles underlying membrane protein structure. Membrane protein assembly processes are both diverse and highly evolved, attesting to the fact that strongly hydrophobic segments in proteins need to be guarded against and contained by tightly regulated processes in order not to wreak havoc on the cell.
The mechanism of capacitative calcium entry is one of themajor unsolved questions in the field of calcium signalling.Cells derive their signal Ca2+from two separate sources. Itcan be released from an internal reservoir, the endoplasmicreticulum (ER), but once this is depleted Ca2’ is drawn infrom the outside. Jim Putney was one of the first to recognizethat the signal to recruit external Ca” comes from the ERand he coined the term capacitive calcium entry (CCE). Byanalogy with an electrical capacitor, the fully charged ER isquiescent but, once it loses its Ca”, it sends a message tochannels in the membrane to induce a Calcium-Release-Activated-Calcium current (ICRAC).It is highly appropriate forPutney to have written this book because he has been at thecentre of research into this puzzling phenomenon. Althoughsomewhat subjective at times, Putney has written a wonder-fully detailed account of how CCE was discovered. He thengoes on to define the main properties of this entrymechanism which clearly establishes the physiologicalcontext in which the missing messenger must operate. Hisbook could be compared to an Agatha Christie detectivenovel and retitled ‘The Search for the Missing Messenger’.Like all good detective stories, Putney begins by setting thescene with a detailed analysis of the key players. In responseto external stimuli, such as hormones, cells generate themessenger inositol trisphosphate ( InsP,) which diffuses to theER where it binds to InsP, receptors which have the channelsthat release stored Ca”. Once the stores empty, the CCEmechanism kicks in when the ER transmits a signal thattravels back to activate entry channels in the plasmamembrane. Here is where the mystery begins because wedon’t know the messenger mechanism. Even the target of thismessenger, the CRAC channels in the membrane, areunknown. There is no shortage of suspects because many ofthe workers in this field, including myself, have put forwardtheir favourite candidates.
A sample of 34 hydrophobia core-segments from known signal sequences have been analysed with regard to the mean hydrophobicity at each position in the segment. The results show that these core-segments are not uniformly hydrophobic; instead, the more hydrophobic residues are concentrated around the midpoints of the segments. A similar analysis carried out on a sample of 12 membrane-spanning segments from transmembrane proteins indicates that the hydrophobic residues are much more evenly distributed along the segments in this case.
No abstract is provided for this article.
Membrane proteins perform many of the essential functions required for life. They are often the targets of medicinal drugs and have many potential uses in biotechnological processes. Therefore our ability to understand them and manipulate their functions is both important and necessary to enable protein engineers to create ‘designer’ membrane proteins (that is, proteins designed to have desired properties).
No abstract is provided for this article.
Signal peptidases remove targeting peptides from pre-proteins and play central roles in the secretory pathway, as well as in the delivery of proteins to the mitochondrial intermembrane space and to the lumen of thylakoids. The catalytic mechanism of pre-protein cleavage has long been an enigma, but recent data from site-directed mutageneis and sequence alignment studies suggest that signal peptidases may constitute a new type of serine protease, mechanistically related to the β-lactamases.
We have shown previously that the 100-residue-long periplasmic N-terminal tail of the <i>Escherichia coli</i> inner membrane protein ProW can be translocated across the inner membrane in a <i>sec</i>-independent manner and that its translocation is blocked by the introduction of three positively charged residues near its C-terminal end (Whitley, P., Zander, T., Ehrmann, M., Haardt, M., Bremer, E., and von Heijne, G.(1994) <i>EMBO J.</i> 13, 4653-4661). We have now further analyzed the requirements for translocation of the N-terminal tail and found that the introduction of even a single arginine can block translocation. Position-specific differences in the effects on translocation of arginine insertions suggest that the C-terminal end of the N-terminal tail is more critical for translocation than the central and N-terminal regions. We also show that the N-terminal tail is translocated in a truncation mutant where a stop codon is placed immediately after the first transmembrane segment, provided that the transmembrane segment is flanked on its C-terminal end by positively charged residues. Thus, <i>sec</i>-independent translocation of a relatively large domain can be induced by a translocation signal located at the extreme C terminus of a protein.
This folder contains the raw data that was used for quantifications (and the quantifications in an Excel sheet). The raw data (.txt files called EasyQuant files) was extracted from autoradiographs of SDS-PAGE gels using the ImageGauge software from Fuji (associated with the Fuji gel scanner). This machine has since been discontinued (these data were collected 2014-2016). The .txt files were imported into EasyQuant and fitted to a Gaussian distribution automatically by the software (software developed in the Gunnar von Heijne lab by Dr. Rickard Hedman), and the Ffl (fraction full length) was calculated.
No abstract is provided for this article.
The eukaryotic cell has a highly developed machinery for routing proteins to their correct intra- or extra-cellular locations. Short stretches of amino acids ("signal peptides") serve as address labels that are recognized by receptors on the surface of the appropriate organelle, where-upon translocation across one or more membranes ensues. Premature folding of the nascent protein is prevented by cytoplasmic "chaperones", i.e. proteins that bind to unfolded or partially folded nascent chains. After import into the organelle, other chaperones are required for catalyzing the final folding of the protein, and for promoting its correct assembly into oligomeric protein complexes. Transport along the secretory pathway (endoplasmic reticulum to Golgi to trans-Golgi to plasma membrane) is mediated by vesicles that bud from a donor compartment and fuse with an acceptor compartment.
No abstract is provided for this article.
To study the sequence requirements for addition of O-linked N-acetylgalactosamine to proteins, amino acid distributions around 174 O-glycosylation sites were compared with distributions around non-glycosylated sites. In comparison with non-glycosylated serine and threonine residues, the most prominent feature in the vicinity of O-glycosylated sites is a significantly increased frequency of proline residues, especially at positions -1 and +3 relative to the glycosylated residues. Alanine, serine and threonine are also significantly increased. The high serine and threonine content of O-glycosylated regions is due to the presence of clusters of several closely spaced glycosylated hydroxy amino acids in many O-glycosylated proteins. Such clusters can be predicted from the primary sequence in some cases, but there is no apparent possibility of predicting isolated O-glycosylation sites from primary sequence data.
