Plasma membrane specialization at the discharge site of the excretory poreless vacuole of the ciliate Euplotes raikovi

The contractile vacuole of Euplotes raikovi consists basically of a membrane-delimited cistern that has no direct communication with the exterior of the cell. Contiguous alveoli interpose between the cisterna and the plasma membrane and probably receive the cisternal contents before being expelled. The area of the plasma membrane that interacts with the outer membrane of the alveoli overlying the cistern has been found to carry a special region of intramembranous particles organized in 100-200 strands, showing varied sizes and a tendency to align parallel with one another. The region is also characterized by a smooth central core and by characteristic deformations that have been interpreted as focal sites of discharge of the contractile vacuole.     

Nature of the phosphorylated 26 000 molecular weight protein extracted from dog thyroid with contractile proteins

Dog thyroid contractile proteins are characterized by their ATPase activity at high KCl concentration. In the presence fo Ca²⁺, 80 nmol ATP are hydrolyzed per min per mg protein. This Ca²⁺-ATPase activity is inhibited by Mg²⁺ but not influenced by sodium azide.The 26 000 molecular weight protein which is present in thyroid contractile protein preparations and the phosphorylation of which is stimulated by thyroid stimulating hormone (TSH) is suggested to be identical to the lysine-rich histones (H₁). Indeed, radioactive thyroid H₁ histones added to unlabelled thyroid slices copurify with the contractile proteins and migrate at the same level as the 26 000 molecular weight protein when submitted to electrophoresis in polyacrylamide sodium dodecyl sulfate gels of different acrylamide concentrations.     

Distribution of alkaline phosphatase in vegetative dictyostelium cells in relation to the contractile vacuole complex

The structure of the contractile vacuole complex of Dictyostelium discoideum has long been a subject of controversy. A model that originated from the work of John Heuser and colleagues described this osmoregulatory organelle as an interconnected array of tubules and cisternae the membranes of which are densely populated with vacuolar proton pumps. A conflicting model described this same organelle as bipartite, consisting of a pump-rich spongiome and a pump-free bladder, the latter membranes being identified by their alkaline phosphatase activity. In the present study we have employed an antiserum specific for Dictyostelium alkaline phosphatase to examine the distribution of this enzyme in vegetative cells. The antiserum labels puncta, probably vesicles, that lie at or near the plasma membrane and are sometimes, but only rarely, enriched near contractile vacuole membranes. We conclude that alkaline phosphatase is not a suitable marker for contractile vacuole membranes. We discuss these results in relation to the two models of contractile vacuole structure and suggest that all data are consistent with the first model.     

How are tonoplast proteins degraded?

Protein turnover is fundamental both for development and cellular homeostasis. The mechanisms responsible for the turnover of integral membrane proteins in plant cells are however still largely unknown. Recently, considerable attention has been devoted to the degradation of plasma membrane proteins. We have now studied the turnover of a tonoplast protein, the potassium channel TPK1, in fully differentiated Arabidopsis leaf cells and showed that its degradation occurs upon internalization into the vacuole. Here, we discuss the possible mechanisms and triggering events involved.     

Phosphorylation of phospholipase B in the plasma membrane of Saccharomyces cerevisiae

Abstract Plasma membrane vesicles from Saccharomyces cerevisiae were incubated with [γ-³²P]ATP. Several phosphorylated protein bands were separated by LiDS polyacrylamide gel electrophoresis. One of these bands with an apparent M _r of 145 000 was identified by immunoprecipitation as a membrane-bound phospholipase.     

A myristoyl/phosphoserine switch controls cAMP-dependent protein kinase association to membranes

The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

Multiple Vacuoles in Plant Cells: Rule or Exception?

It is generally accepted that plant cells can contain multiple vacuoles with different functions, for example lytic vacuoles with lysosome-like properties and protein storage vacuoles for reserve accumulation. Recent data call into question the generality of this theory. In this study, we review the published evidence for the existence of multiple vacuoles. We conclude that the multivacuole hypothesis is valid for a number of cases, but care should be taken before assuming that it applies universally.     

Protein sorting and expression of a unique soybean cotyledon protein,GmSBP, destined for the protein storage vacuole

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and -conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, -conglycinin (/ subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.     

Mycobacterium Leprae Binds to a 25-KDa Phosphorylated Glycoprotein of Human Peripheral Nerve

Mycobacterium leprae, the causative agent of leprosy, specifically invades and destroys the peripheral nerve, which results in the main clinical manifestation of the disease. Little is known about the bacteria—nerve protein interaction. We show in the present work that M leprae binds to a 25 kDa glycoprotein from human peripheral nerve. This protein is phosphorylatable and it binds to lectins which have alpha-mannose specificity. This M leprae-protein interaction could be of importance in the pathogenesis of leprosy.     

Monoclonal Antibody to a Bacterial Endonuclear Symbiont Holospora Cross Reacts with Proteins of Contractile Vacuole Radial Canals of Paramecium Species

ABSTRACT A monoclonal antibody (mAb) IR-2-1 was raised against a 67-kDa protein purified from the macronucleus-specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67-kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR-2-1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa-free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air-dried P. caudatum. These results suggest that the 67-kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule-specific mAb, DS-1, the antigens for IR-2-1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium. Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.     

The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series

As a lysosomal/vacuolar degradative pathway that is conserved in eukaryotic organisms, autophagy mediates the turnover of long-lived proteins and excess or aberrant organelles. The main characteristic of autophagy is the formation of a double-membrane vesicle, the autophagosome, which envelops part of the cytoplasm and delivers it to the lysosome/vacuole for breakdown and eventual recycling of the degradation products. Among the approximately 30 autophagy-related (Atg) genes identified so far, there are two ubiquitin-like proteins, Atg12 and Atg8. Analogous to ubiquitination, Atg12 is conjugated to Atg5 by Atg7--an E1-like protein--and Atg10--an E2-like protein. Similarly, Atg7 and Atg3 are the respective E1-like and E2-like proteins that mediate the conjugation of Atg8 to phosphatidylethanolamine. Both Atg12-Atg5 and Atg8 localize to the developing autophagosome. The Atg12-Atg5 conjugate facilitates the lipidation of Atg8 and directs its correct subcellular localization. Atg8-phosphatidylethanolamine is probably a scaffold protein that supports membrane expansion and the amount present correlates with the size of autophagosomes.     

Application of SVM to predict membrane protein types

As a continuous effort to develop automated methods for predicting membrane protein types that was initiated by Chou and Elrod (PROTEINS: Structure, Function, and Genetics, 1999, 34, 137-153), the support vector machine (SVM) is introduced. Results obtained through re-substitution, jackknife, and independent data set tests, respectively, have indicated that the SVM approach is quite a promising one, suggesting that the covariant discriminant algorithm (Chou and Elrod, Protein Eng. 12 (1999) 107) and SVM, if effectively complemented with each other, will become a powerful tool for predicting membrane protein types and the other protein attributes as well.     

Ethanol induced alterations in plasma membrane protein phosphorylation of neurons and astrocytes

The endogenous protein phosphorylation patterns in plasma membranes of bulk isolated neurons and astroglia from control and chronic ethanol treated rats have been investigated. Chronic ethanol treatment resulted in increased phosphorylation of specific proteins with molecular weights 116, 63 and 60 kDa in both neurons and astrocytes. These proteins were further resolved by 2-DE and the analysis suggested an increased phosphorylation of 10–15 proteins, of which 116 kDa protein is phosphorylated to a higher extent by ethanol. Further, decreased phosphorylation was noticed in D-95 and D-63 proteins in neurons and D-78 and D-54 proteins in astrocytes. Alkali stability experiments for identifying the phosphoamino acid involved in phosphorylation of 116, 63 and 60 kDa proteins suggested that tyrosine and threonine are not involved and probably serine is the likely site for phosphorylation during chronic ethanol treatment. The phosphorylation of specific membrane proteins during chronic ethanol treatment might contribute to ethanol evoked cellular dysfunction.