Nils Johnsson

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.

Ubiquitin-assisted dissection of protein transport across membranes

We describe a new way to analyze targeting in protein translocation. A fusion in which ubiquitin (Ub) is positioned between a signal sequence and a reporter domain is cleaved by Ub‐specific proteases (UBPs) in the cytosol unless the fusion can ‘escape’ into a compartment such as the endoplasmic reticulum (ER). The critical step involves rapid folding of the newly formed Ub moiety, which precludes its translocation and makes possible its cleavage by UBPs. However, if a sufficiently long spacer is present between the signal sequence and Ub, then by the time the Ub polypeptide emerges from the ribosome, the latter is already docked at the transmembrane channel, allowing the translocation of both the Ub and reporter domains of the fusion into the ER. We show that Ub fusions can be used as in vivo probes for kinetic and stochastic aspects of targeting in protein translocation, for distinguishing directly between cotranslational and posttranslational translocation, and for comparing the strengths of different signal sequences. This method should also be applicable to non‐ER translocation.

Pex10p links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome

The protein import machinery of the peroxisome consists of many proteins, collectively called the peroxins. By applying the split-ubiquitin technique we systematically tested the pair-wise interactions between the Nub- and Cub-labeled peroxins for the first time in the living cells of the yeast Saccharomyces cerevisiae. We found that Pex10p plays a central role in the protein interaction network by connecting the ubiquitin conjugation enzyme Pex4p to the other members of the protein import machinery. A yeast strain harboring a deletion of PEX3 enabled us to estimate the influence of the peroxisomal membrane on the formation of a subset of the investigated protein-protein interactions.

A fusion of disciplines: Chemical approaches to exploit fusion proteins for functional genomics

A review of recent developments in the labeling of fusion proteins with synthetic mols. in the living cell or in complex mixts. The most commonly used tags are presented and then focuses on a new class of tags that are not limited to their genetically encoded function but rather serves as general acceptors for synthetic mols. The tetracysteine tag, which reversibly binds to biarsenical compds., and the human O6-alkylguanine-DNA alkyltransferase tag, which is irreversibly alkylated by benzylguanine derivs., are prototypes of this approach. Both provide the resp. fusion protein with functionalities that can not be genetically encoded and thereby complement the properties of the traditional fusion proteins. [on SciFinder (R)]

Protein chemistry on the surface of living cells

The interplay between carbohydrates, lipids, and proteins determines the stability and flexibility as well as the adhesive and responsive features of the surfaces of all cells. The molecular understanding of the interactions among and between the different classes of these biomolecules is rudimentary at best, a lack of suitable experimental methods being the major reason. Here we discuss a new approach for the specific labeling of fusion proteins of carrier proteins with synthetic compounds on cell surfaces and describe how this approach can be used to investigate the properties of the labeled molecules.

Split‐Ubiquitin and the Split‐Protein Sensors: Chessman for the Endgame

The understanding of cellular biology requires a complete, quantitative, and dynamic description of the protein interactions inside the cell. Most of the known interactions were discovered very recently through the use of high-throughput techniques. Hence knowledge about the majority of these interactions hardly exceeds an awareness of their pure existence. The reasons for the difficulty in overcoming this lack of knowledge quickly are partly technical. Citing yeast as a representative example, the majority of the known physical connections between its proteins were derived from co-precipitation studies and two hybrid screens. 2] Both methods investigate proteins in a non-native environment, a condition that makes the straightforward integration of these data into the cellular framework difficult. As a consequence, much weight is now put on identifying and characterizing the interactions of proteins in their natural environments. 4] Casually referred to as “the endgame of protein biochemistry” this endeavor is driven by the development of new and the refinement of existing technologies. Split-ubiquitin (splitUb) is the founding member of a class of analytical tools named split-protein sensors (alternatively referred to as protein fragment complementation assay, PCA) that, based on a common principle, allows measurement of protein interactions and other features of proteins in living cells. Over the years, the application of this common principle to different sensor proteins gave rise to a rich spectrum of new techniques that diverge in their experimental output and their applicability to different cell types or subcellular structures. By focusing on split-Ub we will introduce the properties of these systems and their latest applications.

Protein kinase CK2 phosphorylates Sec63p to stimulate the assembly of the endoplasmic reticulum protein translocation apparatus

The heterotetrameric Sec62/63 complex associates with the heterotrimeric Sec61 complex to form the heptameric Sec complex. This complex is necessary and sufficient for post-translational protein translocation across the membrane of the endoplasmic reticulum. We show that Sec63p is phosphorylated at its C-terminal domain by the protein kinase CK2 and that this phosphorylation strengthens the interaction between the cytosolic domains of Sec63p and Sec62p. Exchanging either threonine 652 or threonine 654 against the nonphosphorylatable alanines in Sec63p impairs the binding to Sec62p and interferes with the efficient translocation of proteins across the membrane of the endoplasmic reticulum. These findings show that phosphorylation of Sec63p is required for tightly recruiting the putative signal-sequence-binding subunit Sec62p to the Sec complex.

A constraint network of interactions: protein-protein interaction analysis of the yeast type II phosphatase Ptc1p and its adaptor protein Nbp2p.

We used a generally applicable strategy to collect and structure the protein interactions of the yeast type II protein phosphatase Ptc1p and its binding partner Nbp2p. The procedure transformed primary unstructured protein interaction data into an ensemble of alternative interaction states. Certain combinations of proteins are allowed in different network configurations. Nbp2p serves as the network hub and brings seven kinases in close contact to Ptc1p. As a consequence, the deletion of NBP2 affects several cellular processes including organelle inheritance and the responses to mating hormone, cell wall stress and high osmolarity; it also impairs the proper execution of the morphogenetic program. Our constraint interaction map provides a basis for understanding a subset of the observed phenotypes and assigns the Ptc1p–Nbp2p module a role in synchronizing the associated kinases during the cell cycle.

alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells.

Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that α2β1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, α2β1 engagement alters assembly of mature focal adhesions by α5β1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of α5β1 to signal for GTP loading of Rac is preserved, the joint engagement of α2β1 interferes with membrane anchorage of Rac. Adapting the ‘split-ubiquitin’ sensor to screen for membrane-proximal α2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by α2β1 integrin. Altogether, our data establish that α2β1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.

Septin rings act as a template for myosin higher-order structures and inhibit redundant polarity establishment.

Summary The mechanisms of the coordinated assembly and disassembly of the septin/myosin ring is central for the understanding of polar growth and cytokinesis in yeast and other organisms. The septin- and myosin-binding protein Bni5p provides a dual function during the formation and disassembly of septin/myosin rings. Early in the cell cycle, Bni5p captures Myo1p at the incipient bud site and actively transforms it into higher-order structures. Additionally, Bni5p stabilizes the septin/myosin ring and is released from the septins shortly before the onset of cytokinesis. If this Bni5p dissociation from the septins is artificially prevented, ring disassembly is impaired and the untimely appearance of septin/myosin ring is induced. The prematurely formed septin/myosin rings delay the establishment of a new polarity axis and the progression into a new cell cycle. This observation suggests a negative feedback between septin/myosin ring formation and polarity establishment that might help to guarantee the singular assembly of this structure and the synchronization of its formation with the cell cycle.