Determining the subcellular localization of a protein is an important first step toward understanding its function. Here, we describe the properties of three well-known N-terminal sequence motifs directing proteins to the secretory pathway, mitochondria and chloroplasts, and sketch a brief history of methods to predict subcellular localization based on these sorting signals and other sequence properties. We then outline how to use a number of internet-accessible tools to arrive at a reliable subcellular localization prediction for eukaryotic and prokaryotic proteins. In particular, we provide detailed step-by-step instructions for the coupled use of the amino-acid sequence-based predictors TargetP, SignalP, ChloroP and TMHMM, which are all hosted at the Center for Biological Sequence Analysis, Technical University of Denmark. In addition, we describe and provide web references to other useful subcellular localization predictors. Finally, we discuss predictive performance measures in general and the performance of TargetP and SignalP in particular.

/pdf/locating-proteins-in-the-cell-using-targetp-signalp-and-39i9pd64rh.pdf

Locating proteins in the cell using TargetP, SignalP and related tools

http://people.umass.edu/~bioch623/623/Second.Section/17.%20JB.BG.Cell.04.review.pdf

The Mechanisms of Vesicle Budding and Fusion

Steroidogenesis entails processes by which cholesterol is converted to biologically active steroid hormones. Whereas most endocrine texts discuss adrenal, ovarian, testicular, placental, and other steroidogenic processes in a gland-specific fashion, steroidogenesis is better understood as a single process that is repeated in each gland with cell-type-specific variations on a single theme. Thus, understanding steroidogenesis is rooted in an understanding of the biochemistry of the various steroidogenic enzymes and cofactors and the genes that encode them. The first and rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone by a single enzyme, P450scc (CYP11A1), but this enzymatically complex step is subject to multiple regulatory mechanisms, yielding finely tuned quantitative regulation. Qualitative regulation determining the type of steroid to be produced is mediated by many enzymes and cofactors. Steroidogenic enzymes fall into two groups: cytochrome P450 enzymes and hydroxysteroid dehydrogenases. A cytochrome P450 may be either type 1 (in mitochondria) or type 2 (in endoplasmic reticulum), and a hydroxysteroid dehydrogenase may belong to either the aldo-keto reductase or short-chain dehydrogenase/reductase families. The activities of these enzymes are modulated by posttranslational modifications and by cofactors, especially electron-donating redox partners. The elucidation of the precise roles of these various enzymes and cofactors has been greatly facilitated by identifying the genetic bases of rare disorders of steroidogenesis. Some enzymes not principally involved in steroidogenesis may also catalyze extraglandular steroidogenesis, modulating the phenotype expected to result from some mutations. Understanding steroidogenesis is of fundamental importance to understanding disorders of sexual differentiation, reproduction, fertility, hypertension, obesity, and physiological homeostasis.

The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders.

The 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. A striking feature of the 14-3-3 proteins is their ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. This plethora of interacting proteins allows 14-3-3 to play important roles in a wide range of vital regulatory processes, such as mitogenic signal transduction, apoptotic cell death, and cell cycle control. In this review, we examine the structural basis for 14-3-3-ligand interactions, proposed functions of 14-3-3 in various signaling pathways, and emerging views of mechanisms that regulate 14-3-3 actions.

14-3-3 Proteins: Structure, Function, and Regulation

In contrast to such “shocks” for which the genome is unprepared, are those a genome must face repeatedly, and for which it is prepared to respond in a programmed manner. Examples are the “heat shock” responses in eukaryotic organisms, and the “SOS” responses in bacteria. Each of these initiates a highly programmed sequence of events within the cell that serves to cushion the effects of the shock. Some sensing mechanism must be present in these instances to alert the cell to imminent danger, and to set in motion the orderly sequence of events that will mitigate this danger. The responses of genomes to unanticipated challenges are not so precisely programmed. Nevertheless, these are sensed, and the genome responds in a discernible but initially unforeseen manner. Barbara McClintock–Nobel lecture , 8 December 1983 These prophetic words by Barbara McClintock eloquently capture the essence of the heat shock response. At the time these words were written, it was known that all organisms shared a common response to physiological stress. Some of the genes encoding heat shock proteins had just been cloned and the basis of heat shock gene regulation was in the early stages of investigation. However, the function of the heat shock response and the role of heat shock proteins were still a mystery. This mystery slowly began to unravel, and by the 1980s, some information on the function of the heat shock proteins as proteases or unfolded polypeptide-binding proteins had already accumulated. These early stages of elucidation of the biochemical...

https://epub.ub.uni-muenchen.de/7685/1/7685.pdf

The Biology of heat shock proteins and molecular chaperones.

Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20.

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH2-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)–induced translation arrest and photo–cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH2-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

https://rupress.org/jcb/article-pdf/151/2/277/1294543/0003112.pdf

Characterization of the Signal That Directs Tom20 to the Mitochondrial Outer Membrane

Mitochondrial precursor proteins with basic targeting signals may be transported across the outer membrane by sequential binding to acidic receptor sites of increasing affinity. To test this 'acid chain' hypothesis, we assayed the interaction of mitochondrial precursors with three acidic receptor domains: the cytosolic domain of Tom20 and the intermembrane space domain of Tom22 and Tim23. The apparent affinity and salt resistance of precursor binding increased in the order Tom20<Tom22 (internal)<Tim23. Precursor binding to the three acidic receptor domains and to the pure cytosolic domain of Tom70 was inhibited by excess targeting peptide, but not by an equally basic control peptide. In this membrane-free and defined system, a precursor pre-bound to the Tom70 or Tom20 domain was transferred efficiently to the Tim23 domain. Transfer was stimulated by the internal Tom22 domain and was much less efficient in the reverse direction. Precursors destined for the outer membrane bound only to Tom20, but not to the internal Tom22 or the Tim23 domain, and a precursor destined for the inner membrane bound only to the Tom20 and the internal Tom22 domain, but not to the Tim23 domain. These results suggest that specific and sequential binding of a targeting signal to strategically situated acidic receptors delivers a precursor across the outer membrane and contributes to intramitochondrial sorting of imported proteins.

/pdf/interaction-of-mitochondrial-targeting-signals-with-acidic-12vzk2nnsf.pdf

Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid chain' hypothesis

The signal sequence of secretory proteins and the signal-anchor sequence of type II membrane proteins initiate the translocation of the following polypeptide segments, whereas the signal-anchor sequence of cytochrome P-450-type membrane proteins mediates the membrane insertion of the polypeptide via a signal-recognition particle-dependent mechanism but does not lead to the translocation of the following C-terminal sequences. To establish the structural requirements for the function of signal and signal-anchor sequences, we constructed chimeric proteins containing artificial topogenic sequences in which the N-terminal net charge and the length of the hydrophobic segment were systematically altered. Utilizing an in vitro translation-translocation system, we found that hydrophobic segments consisting of 7-10 leucine residues functioned as signal sequences whereas segments with 12-15 leucine residues showed different topogenic functions, behaving as signal sequences or P-450-type signal-anchor sequences, depending on the N-terminal charge. From these observations, we propose that the function of N-terminal topogenic sequences depends on a balance between the N-terminal charge and the length of the following hydrophobic segment.

https://www.pnas.org/content/pnas/89/1/16.full.pdf

Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge

We have reconstituted the initial steps of mitochondrial protein import with a purified precursor protein, a purified, ATP-dependent, cytosolic chaperone selective for mitochondrial precursors (mitochondrial import stimulating factor; MSF), and either intact mitochondria or intact or solubilized mitochondrial outer membranes. We show that the precursor-MSF complex first binds to the Mas37p/Mas70p subunits of the mitochondrial import receptor. After ATP-dependent release of MSF, the precursor is transferred from Mas37p/Mas70p to the Mas20p/Mas22p subunits of the receptor, and finally delivered to the import channel in the outer membrane. Import in the absence of the MSF bypasses Mas37p/Mas70p. The ATP-mediated transfer of a precursor from MSF to specific subunits of the import receptor is similar to the GTP-mediated transfer of precursors from the signal recognition particle to its receptor on the endoplasmic reticulum.

Katsuyoshi Mihara

Papers

Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20.

Characterization of the Signal That Directs Tom20 to the Mitochondrial Outer Membrane

Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid chain' hypothesis

Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge

Reconstitution of the initial steps of mitochondrial protein import