The E-Syt3 cleavage and traffic uncovers the primordial cisterna, a new organelle that mothers the lipid droplets in the adipocyte.

Extended synaptotagmins are endoplasmic reticulum proteins consisting of an SMP domain and multiple C2 domains that bind phospholipids and Ca2+ . E-Syts create contact junctions between the ER and plasma membrane (PM) to facilitate the exchange of glycerophospholipids between the apposed membranes. We find in the differentiating adipocyte that the E-Syt3 carboxyl domain is cleaved by a multi-step mechanism that includes removing the C2C domain. Confocal and live-cell time-lapse studies show that truncated E-Syt3ΔC2C, as well as endogenous E-Syt3 and the coat protein PLIN1, target the LDs from an annular, single giant ER cisterna. Inhibition of the proteasome blocks the proteolytic cleavage of Esyt3 and E-Syt3ΔC2C and causes the E-Syt3ΔC2C retention in the giant cisterna. The Esyt3 and PLIN1 distributions and LDs biogenesis show that the primordial cisterna, as we call it, is the birth and nurturing site of LDs in the adipocyte. Isoproterenol-induced lipolysis results in loss of cytoplasmic LDs and reappearance of the primordial cisterna. Electron microscopy and 3D-electron tomography studies show that the primordial cisterna consists of a tightly packed network of varicose tubules with extensively blistered membranes. Rounds of homotypic fusions from nascent to mature LDs play a central role in LD growth. The knockdown of E-Syt3 inhibits LD biogenesis. The identification of the primordial cisterna, an organelle that substitutes the randomly scattered ER foci that mother the LDs in non-adipose cells, sets the stage for a better understanding of LD biogenesis in the adipocyte.

Basolateral sorting and transcytosis define the Cu+-regulated translocation of ATP7B to the bile canaliculus.

The Cu(+) pump ATP7B plays an irreplaceable role in the elimination of excess Cu(+) by the hepatocyte into the bile. The trafficking and site of action of ATP7B are subjects of controversy. One current proposal is that an increase in intracellular Cu(+) results in the translocation of ATP7B to the lysosomes and excretion of excess Cu(+) through lysosomal-mediated exocytosis at the bile canaliculus. Here, we show that ATP7B is transported from the trans-Golgi network (TGN) to the bile canaliculus by basolateral sorting and endocytosis, and microtubule-mediated transcytosis through the subapical compartment. Trafficking ATP7B is not incorporated into lysosomes, and addition of Cu(+) does not cause relocalization of lysosomes and the appearance of lysosome markers in the bile canaliculus. Our data reveal the pathway of the Cu(+)-mediated transport of ATP7B from the TGN to the bile canaliculus and indicates that the bile canaliculus is the primary site of ATP7B action in the elimination of excess Cu(.)

DKWSLLL, a versatile DXXXLL-type signal with distinct roles in the Cu(+)-regulated trafficking of ATP7B.

In the liver, the P-type ATPase and membrane pump ATP7B plays a crucial role in Cu(+) donation to cuproenzymes and in the elimination of excess Cu(+). ATP7B is endowed with a COOH-cytoplasmic (DE)XXXLL-type traffic signal. We find that accessory (Lys -3, Trp -2, Ser -1 and Leu +2) and canonical (D -4, Leu 0 and Leu +1) residues confer the DKWSLLL signal with the versatility required for the Cu(+)-regulated cycling of ATP7B between the trans-Golgi network (TGN) and the plasma membrane (PM). The separate mutation of these residues caused a disruption of the signal, resulting in different ATP7B distribution phenotypes. These phenotypes indicate the key roles of specific residues at separate steps of ATP7B trafficking, including sorting at the TGN, transport from the TGN to the PM and its endocytosis, and recycling to the TGN and PM. The distinct roles of ATP7B in the TGN and PM and the variety of phenotypes caused by the mutation of the canonical and accessory residues of the DKWSLLL signal can explain the separate or joined presentation of Wilson's cuprotoxicosis and the dysfunction of the cuproenzymes that accept Cu(+) at the TGN.

C6orf89 encodes three distinct HDAC enhancers that function in the nucleolus, the golgi and the midbody.

We report here that C6orf89, which encodes a protein that interacts with bombesin receptor subtype-3 and accelerates cell cycle progression and wound repair in human bronchial epithelial cells (Liu et al., 2011, PLoS ONE 6: e23072), encodes one soluble and two type II membrane proteins that function as histone deacetylases (HDAC) enhancers. Soluble 34/64sp is selectively targeted to the nucleolus and is retained in nucleolar organiser regions (NORs) in mitotic cells. Nucleolar 34/64sp is integrated into the ribosomal gene transcription machinery, colocalises and coimmunoprecipitates with the Pol I transcription factor UBF, and undergoes a dramatic relocalisation to the nucleolus upon the arrest of rDNA transcription, protein synthesis and PI3K/mTORC2 signalling. Membrane 42/116mp localises to the Golgi and the midbody, and its controlled ectopic expression provokes the disruption of the Golgi cisternae and hinders the separation of daughter cells and the completion of mitosis. The latter effect is also produced by the microinjection of an affinity-purified amfion antibody. The identification of C60rf89 as a gene that encodes three distinct proteins with the capacity to enhance the activity of histone deacetylases (HDACs) in the nucleolus, the Golgi and the midbody provides new information regarding the components of the acetylome and their capacity to interact with different functional groups in the cell.

The atypical kinase Cdk5 is activated by insulin, regulates the association between GLUT4 and E-Syt1, and modulates glucose transport in 3T3-L1 adipocytes.

Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.

The Cdk5 inhibitor roscovitine strongly inhibits glucose uptake in 3T3-L1 adipocytes without altering GLUT4 translocation from internal pools to the cell surface.

Glucose entry into mammalian cells is facilitated by a family of glucose transport proteins known as GLUTs. Treatment of 3T3-L1 adipocytes with the Cdk5 inhibitor roscovitine strongly inhibits insulin-stimulated/GLUT4-mediated glucose transport. Inhibition of glucose uptake occurs within 2-6 min of the addition of roscovitine and is slowly reversed. The roscovitine treatment interferes with neither the translocation nor the insertion of GLUT4 into the plasma membrane. These studies support recent evidence showing that insulin-stimulated Cdk5 is implicated in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes.

Daxx functions as a scaffold of a protein assembly constituted by GLUT4, JNK1 and KIF5B.

We have previously reported the physical interaction between Daxx, the adaptor protein that mediates activation of the Jun amino-terminal kinase (JNK), and GLUT4, the insulin-dependent glucose transporter, interaction that involves their C-domains. Co-immunoprecipitation and two-hybrid-based protein-protein interaction studies show now that Daxx and GLUT4 interact with JNK1 through D-sites in their NH(2)-(aa 1-501) and large endofacial loop, respectively. Serum deprivation strongly enhances the association of JNK1 with Daxx and dissociates the kinase from GLUT4. SP600125, a potent JNK1 inhibitor, reduces the JNK1 activity associated with GLUT4 and the phosphorylation of two minor GLUT4 species in serum-starved 3T3-L1 adipocytes. In addition, Daxx interacts with kinesin KIF5B through the 6xTPR domain of the kinesin light chain, a domain engaged in the grab hold of protein cargo by kinesin motors that codistribute with JNK. Depletion of Daxx in 3T3-L1 adipocytes provokes the partial translocation of the GLUT4 retained in the GLUT4 storage compartment to endosomes.

ATP7B copper-regulated traffic and association with the tight junctions: copper excretion into the bile.

BACKGROUND & AIMS: The copper transporter ATP7B plays a central role in the elimination of excess copper by the liver into the bile, yet the site of its action remains controversial. The studies reported here examine the correspondence between the site of ATP7B action and distribution and the pathways of copper disposal by the liver. METHODS: Microscopy and cell fractionation studies of polarized Can 10 cells forming long-branched bile canaliculi have been used to study the cellular distribution of ATP7B. Copper excretion into the bile was studied in perfused rat liver. RESULTS: Copper excess provokes a massive download of the ATP7B retained in the trans-Golgi network into the bile canalicular membrane. Furthermore, a stable ATP7B pool is localized to the tight junctions that seal the bile canaliculi. The profile of Cu(64) excretion into the bile by isolated rat livers perfused under one-pass conditions provides evidence of copper excretion by 2 separate mechanisms, transcytosis across the hepatocyte and paracellular transport throughout the tight junctions. CONCLUSIONS: Whereas the ATP7B retained in the trans-Golgi-network is massively translocated to the bile canalicular membrane in response to increased copper levels, a pool of ATP7B associated with the tight junctions remains stable. In situ studies indicate that copper is excreted into the bile by 2 separate pathways. The results are discussed in the frame of the normal and impeded excretion of copper into the bile.

Sorcin links calcium signaling to vesicle trafficking, regulates Polo-like kinase 1 and is necessary for mitosis.

Sorcin, a protein overexpressed in many multi-drug resistant cancers, dynamically localizes to distinct subcellular sites in 3T3-L1 fibroblasts during cell-cycle progression. During interphase sorcin is in the nucleus, in the plasma membrane, in endoplasmic reticulum (ER) cisternae, and in ER-derived vesicles localized along the microtubules. These vesicles are positive to RyR, SERCA, calreticulin and Rab10. At the beginning of mitosis, sorcin-containing vesicles associate with the mitotic spindle, and during telophase are concentrated in the cleavage furrow and, subsequently, in the midbody. Sorcin regulates dimensions and calcium load of the ER vesicles by inhibiting RYR and activating SERCA. Analysis of sorcin interactome reveals calcium-dependent interactions with many proteins, including Polo-like kinase 1 (PLK1), Aurora A and Aurora B kinases. Sorcin interacts physically with PLK1, is phosphorylated by PLK1 and induces PLK1 autophosphorylation, thereby regulating kinase activity. Knockdown of sorcin results in major defects in mitosis and cytokinesis, increase in the number of rounded polynucleated cells, blockage of cell progression in G2/M, apoptosis and cell death. Sorcin regulates calcium homeostasis and is necessary for the activation of mitosis and cytokinesis.

Cdk5, the multifunctional surveyor.

Cyclin-dependent kinases (Cdks) are a family of proline-directed Ser/Thr kinases known for their role in the control of cell cycle progression. In 1992, this family was joined by Cdk5, which is an atypical member in that it uses its own activators and is multifunctional, playing important regulatory roles in multiple cellular functions. Here, we review the structure of Cdk5 and its various mechanisms of activation within the context of the Cdk family. We also address the functional significance of the numerous and varied targets of Cdk5 within pathways that control the organization of the cytoskeleton and focal adhesions, signaling cascades, membrane dynamics and function, cell metabolism, cycle arrest in postmitotic cells, gene transcription and cell survival, all within the context of the adaptation of cells and life systems to changing internal and external environments.

Molecular mechanisms of copper homeostasis.

The transition metal copper (Cu) is an essential trace element for all biota. Its redox properties bestow Cu with capabilities that are simultaneously essential and potentially damaging to the cell. Free Cu is virtually absent in the cell. The descriptions of the structural and functional organization of the metallothioneins, Cu-chaperones and P-type ATPases as well as of the mechanisms that regulate their distribution and functioning in the cell have enormously advanced our understanding of the Cu homeostasis and metabolism in the last decade. Cu is stored by metallothioneins and distributed by specialized chaperones to specific cell targets that make use of its redox properties. Transfer of Cu to newly synthesized cuproenzymes and Cu disposal is performed by the individual or concerted actions of the P-type ATPases ATP7A and ATP7B expressed in tissues. In mammalians liver is the major captor, distributor and excreter of Cu. Mutations in the P-type ATPases that interfere with their functioning and traffic are cause of the life-threatening Wilson (ATP7B) and Menkes (ATP7A) diseases.

Unifying nomenclature for the isoforms of the lysosomal membrane protein LAMP-2.

The present nomenclature of the splice variants of the lysosome-associated membrane protein type 2 (LAMP-2) is confusing. The LAMP-2a isoform is uniformly named in human, chicken, and mouse, but the LAMP-2b and LAMP-2c isoforms are switched in human as compared with mouse and chicken. We propose to change the nomenclature of the chicken and mouse b and c isoforms to agree with that currently used for the human isoforms. To avoid confusion in the literature, we further propose to adopt the use of capital letters for the updated nomenclature of all the isoforms in all three species: LAMP-2A, LAMP-2B, and LAMP-2C.

The insulin-sensitive glucose transporter, GLUT4, interacts physically with Daxx. Two proteins with capacity to bind Ubc9 and conjugated to SUMO1.

In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464-509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-beta (TbetaRII) receptors (1,2 ). The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu(489)-Leu(490)) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.