The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al., 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15°C treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi. TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH2-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH2-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.
The cDNA reported for the gene encoding rat liver propionyl-CoA carboxylase contains a 5' EcoRI fragment of 937 bp which is of bacterial origin and is an artifact of the construction of the library used in these experiments.This quence contains the amino acid sequence for several peptides prepared from purified PCC.This finding raises two questions.First, what is the correct sequence of the 5'-end of the a-PCC cDNA and the corresponding amino-terminal amino acid sequence?Second, how can we reconcile the inconsistencies this discovery introduces into our interpretation of the data from the Northern blot (Fig. 2) and on the mitochondrial import and processing of this construct (Fig. 5)?We are investigating this problem further by cloning an authentic ~l -l e n ~h e-PCC cDNA and will address these questions in a subsequent paper.
Abstract The separation of functional early and late endosomes from other cellular compartments by free‐flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells [1, 2]. Here, using 125 I‐labeled anti‐secretory component antibodies ([ 125 I]SC Ab) and FITC‐labeled asialoorosomucoid (FITC‐ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16°C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20°C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37°C, transcytotic endosomes carrying [ 125 I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC‐ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common ( i.e. early endosomes) and two distinct endosome subpopulations ( i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.
Abstract We have demonstrated that a synthetic peptide corresponding to the rat mitochondrial malate dehydrogenase (mMDH) transit peptide (TP-28) inhibits the binding of pre-mMDH to isolated mitochondria. Synthetic peptides derived from chloroplast transit peptide sequences, which have a similar net charge, did not inhibit import. In addition, this peptide (TP-28) inhibits import of ornithine transcarbamylase, another mitochondrial matrix protein, thus suggesting that common import pathways exist for both mMDH and ornithine transcarbamylase. A smaller synthetic peptide corresponding to residues 1-20 of the mMDH transit peptide (TP-20) also inhibits binding. However, several substitutions for leucine-13 in the smaller peptide relieve import inhibition, thus providing evidence that this neutral residue plays a crucial role in transit peptide binding to the mitochondrial surface. Proteolytic processing of pre-mMDH by a mitochondrial matrix fraction to both the mature and intermediate forms of mMDH was also inhibited by TP-28. The ability of synthetic peptides to inhibit distinct steps in the import of mitochondrial precursor proteins corresponds precisely to their ability to interact with the same components used by transit peptides on intact precursors. Furthermore, inhibition at multiple points along the import pathway reflects the functions of several independent structures contained within transit peptides.
By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking. The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane. To explore this model, we tested for the presence of the putative giantin-p115-GM130 ternary complex. We first mapped p115-binding site in giantin to a 70-amino acid coiled-coil domain at the extreme N terminus, a position that may exist up to 400 nm away from the Golgi membrane. We then generated glutathioneS-transferase (GST) fusion proteins containing either giantin's or GM130's p115 binding site and tested whether such proteins could bind p115 and GM130 or bind p115 and giantin, respectively. Unexpectedly, GST fusions containing either the giantin or the GM130 p115 binding site efficiently bound p115, but the p115 bound to GST-giantin did not bind GM130, and the p115 bound to GST-GM130 did not bind giantin. To explain this result, we mapped the giantin binding site in p115 and found that it is located at the C-terminal acidic domain, the same domain involved in binding GM130. The presence of a single binding site in p115 for giantin and GM130 was confirmed by demonstration that giantin and GM130 compete for binding to p115. These results question a simple tethering model involving a ternary giantin-p115-GM130 complex and suggest that p115-giantin and p115-GM130 interactions might mediate independent membrane tethering events.
ABSTRACT Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1 , encoding fibrocystin (FPC), but Pkhd1 mutant mice fail to express renal cystic disease. In contrast, the renal lesion in Cys1 cpk/cpk ( cpk ) mice with loss of the cystin protein, closely phenocopy ARPKD. Recent identification of patients with CYS1 -related ARPKD prompted the investigations described herein. We analyzed cystin and FPC expression in mouse models ( cpk , rescued- cpk ( r - cpk ), Pkhd1 mutants) and cortical collecting duct (CCD) cell lines (wild type ( wt), cpk) . We found that cystin deficiency led to diminished FPC in both cpk kidneys and CCD cells. In r-cpk kidneys, FPC increased and siRNA of Cys1 in wt CCD cells reduced FPC. Conversely, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and the associated reduction in FPC levels impacted the architecture of the primary cilium, but not ciliogenesis. Similar Pkhd1 mRNA levels in wt, cpk kidneys and CCD cells suggested posttranslational mechanisms directed FPC loss and studies of cellular protein degradation systems revealed selective autophagy as a possible mechanism. Loss of FPC from the NEDD4 E3 ubiquitin ligase complexes caused reduced polyubiquitination and elevated levels of functional epithelial sodium channel (NEDD4 target) in cpk cells. We propose that cystin is necessary to stabilize FPC and loss of cystin leads to rapid FPC degradation. FPC removal from E3-ligase complexes alters the cellular proteome and may contribute to cystogenesis through multiple mechanisms, that include MYC transcriptional regulation.
Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes, including penetration of the plasma membrane, initiation of infection, replication, and suppression of the host's antiviral defenses. In this review, we focus on picornaviruses, a family of positive-strand RNA viruses, and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular-membrane-remodeling proteins and led to the development of models in which the virus used a preexisting cellular-membrane-shaping pathway "as is" for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the proteincentric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears once again that the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multilayered, complex mechanisms that control the membrane organization of a eukaryotic cell remains very limited.
The gene cAMP-responsive element binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with null mutants exhibiting fragile bones, and in human where homozygous CREB3L1 mutations are associated with severe osteogenesis imperfecta. However, the mechanism via which CREB3L1 regulates bone development is not understood in depth. To further probe the role of CREB3L1 in organismal physiology, we used CRISPR-Cas9 genome editing and generated a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to the strong phenotypes observed in mammals, the Creb3l1 deficient fish do not display abnormalities in osteogenesis. Both skeletal morphology and overall bone density appear normal in the mutant fish. Despite this, regeneration of caudal fin post-amputation is affected, with a decrease in overall regenerate and mineralized area. Moreover, the mutant fish exhibit a severe patterning defect, with a significant decrease in bifurcation of the regenerating fin rays and distalization of the bifurcation site. In situ hybridization analysis of expression of genes implicated in bone development showed aberrant patterning of shha and ptch2, hence linking Creb3l1 with the Sonic Hedgehog signaling pathway during fin regeneration. Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during caudal fin regeneration.
