A genomic integration method for the simultaneous visualization of endogenous mRNAs and their translation products in living yeast

Protein localization within cells can be achieved by the targeting and localized translation of mRNA. Yet, our understanding of the dynamics of mRNA targeting and protein localization, and of how general this phenomenon is, is not clear. Plasmid-based expression systems have been used to visualize exogenously expressed mRNAs and proteins; however, these methods typically produce them at levels greater than endogenous and can result in mislocalization. Hence, a method that allows for the simultaneous visualization of endogenous mRNAs and their translation products in living cells is needed. We previously developed a method (m-TAG) to localize endogenously expressed mRNAs in yeast by chromosomal insertion of the MS2 aptamer sequence between the open-reading frame (ORF) and 3' UTR of any gene. Upon coexpression with the MS2 RNA-binding coat protein (MS2-CP) fused with GFP, the aptamer-tagged mRNAs bearing their 3' UTRs are localized using fluorescence microscopy. Here we describe an advanced method (mp-TAG) that allows for the simultaneous visualization of both endogenously expressed mRNAs and their translation products in living yeast for the first time. Homologous recombination is used to insert the mCherry gene and MS2-CP binding sites downstream from any ORF, in order to localize protein and mRNA, respectively. As proof of the concept, we tagged ATP2 as a representative gene and demonstrated that endogenous ATP2 mRNA and protein localize to mitochondria, as shown previously. In addition, we demonstrate that tagged proteins like Hhf2, Vph1, and Yef3 localize to their expected subcellular location, while the localization of their mRNAs is revealed for the first time.     

Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stability

Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N‐linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high‐molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N → A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high‐affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N‐linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N‐linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex.     

A role of GCC88 in the retrograde transport of CI‐M6PR and the maintenance of lysosomal activity

GCC88 is a golgin coiled‐coil protein at the trans‐Golgi (TGN) that functions as a tethering factor for the endosome‐derived retrograde transport vesicles. Here, we demonstrate that GCC88 is required for the endosome‐to‐TGN retrograde transport of the cation‐independent mannose 6‐phosphate receptor (CI‐M6PR). The knockout of GCC88 perturbs the retrieval of CI‐M6PR and decreases its cellular level at the steady state, which causes the improper processing of newly synthesized cathepsin‐D, a lysosomal hydrolase dependent on CI‐M6PR for its delivery to lysosomes. At the whole cell level, the knockout of GCC88 reduces the lysosomal proteolytic capacity but does not impair of the efficiency of autophagy within these cells.     

Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex

The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.     

参与植物膜泡运输的蛋白质家族及功能研究进展

真核细胞的内吞和分泌途径中蛋白质和脂类的运输主要由膜泡运输介导。参与膜泡运输的蛋白质家族包括SNARE蛋白家族、RAB蛋白家族、被膜蛋白复合体、Sec1蛋白家族、Arf蛋白家族。这些蛋白质家族在进化中高度保守,并且在植物中已经鉴定了许多哺乳动物和酵母蛋白的同源物。近年来一些研究发现这些蛋白质不仅仅调节植物细胞的膜泡运输,还影响植物的许多生理活动和功能,例如向重性生长、胞质分裂、激素极性运输、气孔运动以及抗病性等。现主要阐述迄今在植物中研究这五类蛋白质家族功能的最新进展。     

Protein trafficking and maturation regulate intramembrane proteolysis

Intramembrane-cleaving proteases (I-CLiPs) are membrane embedded proteolytic enzymes. All substrates identified so far are also membrane proteins, involving a number of critical cellular signaling as well as human diseases. After synthesis and assembly at the endoplasmic reticulum, membrane proteins are exported to the Golgi apparatus and transported to their sites of action. A number of studies have revealed the importance of the intracellular membrane trafficking in i-CLiP-mediated intramembrane proteolysis, not only for limiting the unnecessary encounter between i-CLiPs and their substrate but also for their cleavage site preference. In this review, we will discuss recent advances in our understanding of how each i-CLiP proteolysis is regulated by intracellular vesicle trafficking. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Differential palmitoylation of the endosomal SNAREs syntaxin 7 and syntaxin 8

Palmitoylation is a posttranslational modification that regulates protein trafficking and stability. In this study we investigated whether the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins syntaxin 7 and syntaxin 8 are modified with palmitate. Using metabolic labeling and site-directed mutagenesis, we show that human syntaxins 7 and 8 are modified with palmitate through a thioester linkage. Palmitoylation is dependent upon cysteine 239 of human syntaxin 7 and cysteine 214 of syntaxin 8, residues that are located on the cytoplasmic face of the transmembrane domain (TMD). Palmitoylation of syntaxin 8 is minimally affected by the Golgi-disturbing agent brefeldin A (BFA), whereas BFA dramatically inhibits palmitoylation of syntaxin7. The differential effect of BFA suggests that palmitoylation of syntaxins 7 and 8 occurs in distinct subcellular compartments. Palmitoylation does not affect the rate of protein turnover of syntaxins 7 and 8 nor does it influence the steady-state localization of syntaxin 8 in late endosomes. Syntaxin 7 actively cycles between endosomes and the plasma membrane. Palmitoylation-defective syntaxin 7 is selectively retained on the plasma membrane, suggesting that palmitoylation is important for intercompartmental transport of syntaxin 7.     

Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca(2+)-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca(2+), a approximately 35-40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca(2+), and the interaction is direct and optimal at 1 microM Ca(2+). Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca(2+)-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.     

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons

J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.     

The rice RING E3 ligase,OsCTR1, inhibits trafficking to the chloroplasts of OsCP12 and OsRP1, and its overexpression confers drought tolerance in Arabidopsis

Plant growth under low water availability adversely affects many key processes with morphological, physiological, biochemical and molecular consequences. Here, we found that a rice gene, OsCTR1, encoding the RING Ub E3 ligase plays an important role in drought tolerance. OsCTR1 was highly expressed in response to dehydration treatment and defense‐related phytohormones, and its encoded protein was localized in both the chloroplasts and the cytosol. Intriguingly, the OsCTR1 protein was found predominantly targeted to the cytosol when rice protoplasts transfected with OsCTR1 were treated with abscisic acid (ABA). Several interacting partners were identified, which were mainly targeted to the chloroplasts, and interactions with OsCTR1 were confirmed by using biomolecular fluorescence complementation (BiFC). Interestingly, two chloroplast‐localized proteins (OsCP12 and OsRP1) interacted with OsCTR1 in the cytosol, and ubiquitination by OsCTR1 led to protein degradation via the Ub 26S proteasome. Heterogeneous overexpression of OsCTR1 in Arabidopsis exhibited hypersensitive phenotypes with respect to ABA‐responsive seed germination, seedling growth and stomatal closure. The ABA‐sensitive transgenic plants also showed improvement in their tolerance against severe water deficits. Taken together, our findings lend support to the hypothesis that the molecular functions of OsCTR1 are related to tolerance to water‐deficit stress via ABA‐dependent regulation and related systems.     

Cell biology of the plant Golgi apparatus

The higher plant Golgi apparatus, comprising many individual stacks of membrane bounded cisternae, is one of the most enigmatic of the cytoplasmic organelles. Not only can the stacks receive material from the endoplasmic reticulum, process it and target it to the correct cellular destination, but they can also synthesise and export complex carbohydrates and lipids and most likely act as one end point of the endocytic pathway. In many cells such processing and sorting can take place while the stacks are moving within the cytoplasm and, remarkably, the organelle manages to retain its structural integrity. This review considers some of the latest data and views on transport both to and from the Golgi and the mechanisms by which such activity is regulated.     

Tetraspanins as regulators of protein trafficking

Small transmembrane proteins of the tetraspanin superfamily are believed to function as the main structural blocks of specialized membrane microdomains (referred to as tetraspanin-enriched microdomains, TERM or TEM). Through a multitude of homotypic and heterotypic interactions, tetraspanins regulate lateral clustering and, consequently, signalling involving adhesion and growth factor receptors as well as costimulatory proteins. The presence of major histocompatibility complex (MHC) I and MHCII molecules in TERM led to suggestion of tetraspanins' involvement in antigen presentation. In addition, certain tetraspanins function as viral co-receptors and may be important for viral egress from infected cells. It has recently become apparent that in addition to their purely structural function as organizers of TERM, tetraspanins also regulate various aspects of trafficking and biosynthetic processing of associated receptors. Here, we review recent studies, which specifically focus on this issue.     

Crystal structure of the S.cerevisiae exocyst component Exo70p

The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.     

Fates of Sec,Tat, and YidC Translocases in Mitochondria and Other Eukaryotic Compartments

Formation of mitochondria by the conversion of a bacterial endosymbiont was a key moment in the evolution of eukaryotes. It was made possible by outsourcing the endosymbiont’s genetic control to the host nucleus, while developing the import machinery for proteins synthesized on cytosolic ribosomes. The original protein export machines of the nascent organelle remained to be repurposed or were completely abandoned. This review follows the evolutionary fates of three prokaryotic inner membrane translocases Sec, Tat, and YidC. Homologs of all three translocases can still be found in current mitochondria, but with different importance for mitochondrial function. Although the mitochondrial YidC homolog, Oxa1, became an omnipresent independent insertase, the other two remained only sporadically present in mitochondria. Only a single substrate is known for the mitochondrial Tat and no function has yet been assigned for the mitochondrial Sec. Finally, this review compares these ancestral mitochondrial proteins with their paralogs operating in the plastids and the endomembrane system.     

Molecular dissection of the Munc18c/syntaxin4 interaction: implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.     

Dominant-negative behavior of mammalian Vps35 in yeast requires a conserved PRLYL motif involved in retromer assembly

The retromer protein complex assists in recycling selected integral membrane proteins from endosomes to the trans Golgi network. One protein subcomplex (Vps35p, Vps26p and Vps29p) combines with a second (Vps17p and Vps5p) to form a coat involved in sorting and budding of endosomal vesicles. Yeast Vps35p (yVps35) exhibits similarity to human Vps35 (hVps35), especially in a completely conserved PRLYL motif contained within an amino-terminal domain. Companion studies indicate that an R(98)W mutation in yVps35 causes defective retromer assembly in Saccharomyces cerevisiae. Herein, we find that the expression of hVps35 in yeast confers dominant-negative vacuolar proenzyme secretion and defective secretory proprotein processing. The mutant phenotype appears to be driven by hVps35 competing with endogenous yVps35, becoming incorporated into defective retromer complexes and causing proteasomal degradation of endogenous Vps26 and Vps29. Increased expression of yVps35 displaces some hVps35 to a 100 000 x g supernatant and suppresses the dominant-negative phenotype. Remarkably, mutation of the conserved R(107)W of hVps35 displaces some of the protein to the 100 000 x g supernatant, slows protein turnover and restores stability of Vps26p and Vps29p and completely abrogates dominant-negative trafficking behavior. We show that hVps35 coprecipitates Vps26, whereas the R(107)W mutant does not. In pancreatic beta cells, the R(107)W mutant shifts hVps35 from peripheral endosomes to a juxtanuclear compartment, affecting both mannose phosphate receptors and insulin. These data underscore importance of the Vps35 PRLYL motif in retromer subcomplex interactions and function.     

The fat controller: roles of palmitoylation in intracellular protein trafficking and targeting to membrane microdomains (Review)

The attachment of palmitic acid to the amino acid cysteine via thioester linkage (S-palmitoylation) is a common post-translational modification of eukaryotic proteins. In this review, we discuss the role of palmitoylation as a versatile protein sorting signal, regulating protein trafficking between distinct intracellular compartments and the micro-localization of proteins within membranes.     

Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) Specifically Interacts with Phospholipid Transfer Protein StarD10 to Facilitate Surfactant Phospholipid Trafficking in Alveolar Type II Cells

Pulmonary surfactant, a mixture of proteins and phospholipids, plays an important role in facilitating gas exchange by maintaining alveolar stability. Saturated phosphatidylcholine (SatPC), the major component of surfactant, is synthesized both de novo and by the remodeling of unsaturated phosphatidylcholine (PC) by lyso-PC acyltransferase 1 (LPCAT1). After synthesis in the endoplasmic reticulum, SatPC is routed to lamellar bodies (LBs) for storage prior to secretion. The mechanism by which SatPC is transported to LB is not understood. The specificity of LPCAT1 for lyso-PC as an acyl acceptor suggests that formation of SatPC via LPCAT1 reacylation is a final step in SatPC synthesis prior to transport. We hypothesized that LPCAT1 forms a transient complex with SatPC and specific phospholipid transport protein(s) to initiate trafficking of SatPC from the endoplasmic reticulum to the LB. Herein we have assessed the ability of different StarD proteins to interact with LPCAT1. We found that LPCAT1 interacts with StarD10, that this interaction is direct, and that amino acids 79–271 of LPCAT1 and the steroidogenic acute regulatory protein-related lipid transfer (START) domain of START domain-containing protein 10 (StarD10) are sufficient for this interaction. The role of StarD10 in trafficking of phospholipid to LB was confirmed by the observation that knockdown of StarD10 significantly reduced transport of phospholipid to LB. LPCAT1 also interacted with one isoform of StarD7 but showed no interaction with StarD2/PC transfer protein.     

Membranous structures transfer cell surface proteins across NK cell immune synapses

Intercellular transfer of cell surface proteins is widespread and facilitates several recently discovered means for immune cell communication. Here, we examined the molecular mechanism for intercellular exchange of the natural killer (NK) cell receptor KIR2DL1 and HLA-C, prototypical proteins that swap between NK cells and target cells. Transfer was contact dependent and enhanced for cells expressing cognate receptor/ligand pairs but did not depend on KIR2DL1 signaling. To a lesser extent, proteins transferred independent from specific recognition. Intracellular domains of transferred proteins were not exposed to the extracellular environment and transferred proteins were removed by brief exposure to low pH. By fluorescence microscopy, transferred proteins localized to discrete regions on the recipient cell surface. Higher resolution scanning electron micrographs revealed that transferred proteins were located within specific membranous structures. Transmission electron microscopy of the immune synapse revealed that membrane protrusions from one cell interacted with the apposing cell surface within the synaptic cleft. These data, coupled with previous observations, lead us to propose that intercellular protein transfer is mediated by membrane protrusions within and surrounding the immunological synapse.