Traffic
CREB‐H and activating transcription factor 6 (ATF6) are transmembrane transcription factors that, in response to endoplasmic reticulum (ER) stress, traffic to the Golgi where they are cleaved by specific proteases, producing the N‐terminal domains that effect appropriate transcriptional responses. We show that unlike in ATF6 whose lumenal tail binds BiP and contains determinants for stress sensing and Golgi transport, in CREB‐H the lumenal tail is not involved in ER retention, not required for Golgi transport and does not bind BiP. The main determinant for CREB‐H ER retention resides in a membrane‐proximal cytoplasmic determinant that is conserved in related members of the CREB‐H family, but lacking in ATF6. We refine requirements within the ER‐retention motif (ERM) and show that ERM‐ve variants exhibited constitutive Golgi localization and constitutive cleavage by the Golgi protease, S1P. The ERM also conferred ER retention on a heterologous protein. Furthermore, deletion of the lumenal tail of CREB‐H had no effect on ER retention of parental CREB‐H or Golgi localization of ERM‐ve variants. Importantly, when the lumenal tail of ATF6 was transferred into an ERM‐ve variant, the chimera was now retained in the ER. Together, these data demonstrate novel and qualitatively distinct mechanisms of trafficking and stress signalling in CREB‐H compared to ATF6....
The stoned proteins, stoned A (STNA) and stoned B (STNB), are essential for normal vesicle trafficking in Drosophila melanogaster neurons, and deletion of the stoned locus is lethal. Although there is a growing body of research aimed at defining the roles of these proteins, particularly for STNB where homologues have now been identified in all multicellular species, their functions and mechanisms...
Argonaute proteins are the effectors of small RNA‐dependent gene‐silencing pathways. In the cytoplasm, they are incorporated into large mobile ribonucleoprotein (RNP) complexes that travel along microtubules. We used a genetic screen to identify the microtubule‐associated motor that interacts with Ago1‐containing RNPs. Here, we report that activity of the kinesin family member Cut7 is important for biogenesis and/or stability of Ago1‐containing RNPs in the cytoplasm. Results from pulldown and coimmunoprecipitation assays indicate that Cut7 interacts with Ago1 as well as its two cognate binding proteins, Dcr1 and Rdp1. Loss of Cut7 activity was associated with increased levels of reverse centromeric transcripts, presumably because of a defect in post‐transcriptional gene silencing. Overexpression of the Ago1‐binding region of Cut7 resulted in loss of microscopic Ago1‐containing RNPs. Together, these results suggest that microtubule motor proteins function in the biogenesis and function of gene‐silencing machinery in the cytoplasm....
Vascular endothelial growth factor A (VEGF‐A)‐induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2‐mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF‐A‐stimulated signaling and endothelial cell migration. Ligand‐stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT‐0 subunit caused differential effects on ligand‐stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF‐A‐induced intracellular signaling to c‐Akt and endothelial nitric oxide synthase (eNOS). VEGF‐A‐stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF‐A‐mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C‐dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF‐stimulated endothelial signaling and cell migration....
Cyclic AMP (cAMP)‐dependent protein kinase A (PKA) is part of the set of signaling proteins that are stably associated to the cytosolic surface of Golgi membranes in mammalian cells. In principle, Golgi‐associated PKA could participate in either signal transduction events and/or the coordination of Golgi transport activities. Here, we show data indicating that although Golgi‐associated PKA is activated fast and efficiently during cell stimulation by an extracellular ligand it does not contribute significantly to cAMP signal transmission to the nucleus. Instead, most of the PKA catalytic subunits C...
Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3‐D electron tomographic study of the endothelial caveolar system in situ. Analysis of large cellular volumes of (high‐pressure frozen, freeze‐substituted and epon‐embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on...
The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau‐Scherrer C, Raposo G, Bonnerot C. Exosomal‐like vesicles are present in human blood plasma. Int Immunol 2005;17:879–887). Extracellular Nef protein has deleterious effects on CD4+ T cells (James CO,...
The mammalian Golgi apparatus consists of individual cisternae that are stacked in a polarized manner to form the compact zones of the Golgi. Several stacks are linked to form a ribbon via dynamic lateral bridges. The determinants required for maintaining the characteristic Golgi structure are incompletely understood. Here, we have characterized p28, a new γ‐subfamily member of p24 membrane proteins...
Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER‐associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H +‐ATPase, render misfolded proteins that are subjected to ERAD. A subset of misfolded PMA1 mutants exhibits a dominant negative effect on yeast growth since, when co‐expressed with the wild...
Stonins are a small family of evolutionarily conserved clathrin adaptor complex AP‐2μ‐related factors that may act as cargo‐specific sorting adaptors in endocytosis and perhaps beyond. Whereas little is known about the localization and function of stonin 1, recent work suggests that stonin 2 serves as a linker between the endocytic proteins AP‐2 and Eps15 and the calcium‐sensing synaptic vesicle (SV) protein synaptotagmin 1. The molecular determinants involved in the recognition of SV cargo by the μ‐homology domain of stonin 2 are evolutionarily conserved from worm to man, thereby
identifying stonin 2 and its invertebrate homologs uncoordinated (UNC)‐41 and stoned B as endocytic adaptors dedicated to the retrieval of surface‐stranded SV proteins, most notably synaptotagmin. In this review, we summarize the current state of knowledge about mammalian stonins with a special focus on the role of stonin 2 in SV recycling at presynaptic nerve terminals....
The role of PIP2 in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP2 with PH‐PLC‐GFP or PIP5KIγ RNAi did not impact on glucose‐stimulated secretion although susceptibility to apoptosis was increased. Over‐expression of PIP5KIγ improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F‐actin, PIP2, transferrin receptor,...
The formation of a primary endocytic vesicle is a dynamic process involving the transient organization of adaptor and scaffold proteins at the plasma membrane. Epsins and Eps15‐like proteins are ubiquitin‐binding proteins that act early in this process. The yeast epsins, Ent1 and Ent2, carry functional ubiquitin‐interacting motifs (UIMs), whereas the yeast Eps15‐like protein, Ede1, has a C‐terminal ubiquitin‐associated (UBA) domain. Analysis of mutants lacking early endocytic adaptors reveals that the ubiquitin‐binding domains (UBDs) of Ent2 and Ede1 are likely to function primarily to mediate protein–protein interactions between components of the early endocytic machinery. Cells that lack epsin and Ede1 UBDs are able to internalize activated, ubiquitinated receptors. Furthermore, under conditions in which epsin UIMs are important for receptor internalization, receptors internalized via both ubiquitin‐dependent and ubiquitin‐independent signals require the UIMs, indicating that UIM function is not restricted to ubiquitinated receptors. Epsin UIMs share function with non‐UBD protein–protein interaction motifs in Ent2 and Ede1, and the Ede1 UBA domain appears to negatively regulate interactions between endocytic proteins. Together, our results suggest that the ubiquitin‐binding domains within the yeast epsin Ent2 and Ede1 are involved in the formation and regulation of the endocytic network....
The digestive vacuole plays an important role in the pathophysiology of the human malaria parasite Plasmodium falciparum. It is a terminal degradation organelle involved in the proteolysis of the host erythrocyte's haemoglobin; it is the site of action of several antimalarial drugs and its membrane harbours transporters implicated in drug resistance. How the digestive vacuole recruits residential proteins is largely unknown. Here, we have investigated the mechanism underpinning trafficking of the chloroquine resistance transporter, PfCRT, to the digestive vacuolar membrane. Nested deletion analysis and site‐directed mutagenesis identified threonine 416 as a functional residue for sorting PfCRT to its site of residence. Mass spectroscopy demonstrated that threonine 416 can be phosphorylated. Further phosphorylation was detected at serine 411. Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane....
Acidic extracellular pH (pHe) has been shown to stimulate peripheral lysosome trafficking, resulting in cathepsin B secretion and tumor invasion. In addition, inhibitors of sodium‐proton exchangers (NHE) such as EIPA, cariporide and s3226, as well as the non‐specific NHE inhibitor, troglitazone (Tro), blocked these changes. In this paper, we report a differential ability of the thiazolidinedione (TZD) family of compounds to induce a time‐dependent retrograde aggregation of lysosomes over the microtubule‐organizing center (MTOC) in tumor cells exposed to acidic pHe. This trafficking event depended on microtubules and the MAP‐Kinase pathway, but was independent of Rho GTPase activity. Expression of shRNA implicated Rab7 in this process, and subcellular fractionation revealed that levels of Rab7, RILP and Erk1/2 were increased on lysosomes purified from cells treated with Tro. In addition, DN‐RILP overexpression studies indicated that this Rab7 effector also played a role in TZD‐induced retrograde trafficking. Tro was able to prevent acidic pHe‐induced cell invasion. Finally, DU145 prostate tumor cells stably over‐expressing WT‐RILP, a condition where lysosomes aggregate to the MTOC in the absence of Tro, did not invade in response to acidic pHe, suggesting that the regulation of lysosome trafficking is an inherently important aspect of tumor cell invasion....
Peroxisomes are unique organelles which display properties of autonomous organelles, as they can multiply by fission of pre‐existing ones. Peroxisomes, however, can also develop from the endoplasmic reticulum (ER). This process has convincingly been shown in peroxisome‐deficient yeast cells, upon reintroduction of the corresponding gene. Whether peroxisomes also are formed from the ER in wild‐type cells that contain peroxisomes is still under debate. Also, the existence of vesicular transport pathways between peroxisomes and the ER is still unresolved....
The downregulation of cell surface receptors by endocytosis is a fundamental requirement for the termination of signalling responses and ubiquitination is a critical regulatory step in receptor regulation. The K5 gene product of Kaposi's sarcoma‐associated herpesvirus is an E3 ligase that ubiquitinates and downregulates several cell surface immunoreceptors, including major histocompatibility complex...
Functional defects in cilia are associated with various human diseases including congenital hydrocephalus. Previous studies suggested that defects in cilia not only disrupt the flow of cerebrospinal fluid (CSF) generated by motile cilia in ependyma lining the brain ventricles, but also cause increased CSF production at the choroid plexus. However, the molecular mechanisms of CSF overproduction by ciliary dysfunction remain elusive. To dissect the molecular mechanisms, choroid plexus epithelial cells (CPECs) were isolated from porcine brain. These cells expressed clusters of primary cilia on the apical surface. Deciliation of CPECs elevated the intracellular cyclic AMP (cAMP) levels and stimulated basolateral‐to‐apical fluid transcytosis, without detrimental effects on other morphological and physiological features. The primary cilia possessed neuropeptide FF (NPFF) receptor 2. In deciliated cells, the responsiveness to NPFF was reduced at nanomolar concentrations. Furthermore, CPECs expressed NPFF precursor along with NPFFR2. An NPFFR antagonist, BIBP3226, increased the fluid transcytosis, suggesting the presence of autocrine NPFF signaling in CPECs for a tonic inhibition of fluid transcytosis. These results suggest that the clusters of primary cilia in CPECs act as a sensitive chemosensor to regulate CSF production....
We analyzed the nuclear import and regulation of the yeast histone variant Htz1 (H2A.Z), and the role of histone chaperones Nap1 and Chz1 in this process. Copurification suggested that Htz1 and H2B dimerized in the cytoplasm prior to import. Like H2B, Htz1 contained a nuclear localization signal (NLS) in its N‐terminus that is recognized by multiple karyopherins (also called importins), indicating...