No abstract

Autophagy delivers cytoplasmic components and organelles to lysosomes for degradation. This pathway serves to degrade nonfunctional or unnecessary organelles and aggregate-prone and oxidized proteins to produce substrates for energy production and biosynthesis. Macroautophagy delivers large aggregates and whole organelles to lysosomes by first enveloping them into autophagosomes that then fuse with lysosomes. Chaperone-mediated autophagy (CMA) degrades proteins containing the KFERQ-like motif in their amino acid sequence, by transporting them from the cytosol across the lysosomal membrane into the lysosomal lumen. Autophagy is especially important for the survival and homeostasis of postmitotic cells like neurons, because these cells are not able to dilute accumulating detrimental substances and damaged organelles by cell division. Our current knowledge on the autophagic pathways and molecular mechanisms and regulation of autophagy will be summarized in this review. We will describe the physiological functions of macroautophagy and CMA in neuronal cells. Finally, we will summarize the current evidence showing that dysfunction of macroautophagy and/or CMA contributes to neuronal diseases. We will give an overview of our current knowledge on the role of autophagy in aging neurons, and focus on the role of autophagy in four types of neurodegenerative diseases, i.e., amyotrophic lateral sclerosis and frontotemporal dementia, prion diseases, lysosomal storage diseases, and Parkinson's disease.

Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli ␤-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. ␤-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150⌬, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150⌬-␤-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar ␤-lactamase activity and K m values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation. INTRODUCTIONTranslocation of newly synthesized precursor proteins into the yeast endoplasmic reticulum (ER) occurs cotranslationally or posttranslationally, depending on the hydrophobicity of the signal peptides (Brodsky and Schekman, 1994;Ng et al., 1996;Rapoport et al., 1996). In the cotranslational pathway, the signal recognition particle binds to the signal peptide emerging from the ribosome, translation halts, and the nascent chain-ribosome complex is targeted to the trimeric Sec61 translocon complex (Sec61p, Sbh1p, and Sss1p) embedded in the ER membrane (Panzner et al., 1995). The polypeptide traverses the aqueous translocon channel simultaneously with elongation, apparently in an extended form, whereafter it adopts its native structure in the ER lumen. For instance, in cotranslational translocation of the Escherichia coli Lep protein into canine microsomes, 65 amino acids bridge the ribosomal P site and the luminal surface of the ER membrane (Whitley et al., 1996). In contrast, posttranslational translocation is signal recognition particle independent. Translation of the polypeptide is completed on free ribosomes, whereafter the preprotein traverses the ER membrane via the translocon complex associated with the Sec62-63 complex (Sec62p, Sec63p, Sec71p, and Sec72p;Deshaies and Schekman, 1989;Rothblatt et al., 1989;Deshaies et al., 1991;Feldheim and Schekman, 1994;Panzner et al., 1995). Because the amino acid sequence primarily dictates the three-dimensional structure of proteins, polypeptides could fold in the cytoplasm unless they were prevented from folding. Depletion of two of the four predominant Hsp70 homologues of the yea...

Sec13p has been thought to be an essential component of the COPII coat, required for exit of proteins from the yeast endoplasmic reticulum (ER). We show herein that normal function of Sec13p was not required for ER exit of the Hsp150 glycoprotein. Hsp150 was secreted to the medium under restrictive conditions in a sec13-1 mutant. The COPII components Sec23p and Sec31p and the GTP/GDP exchange factor Sec12p were required in functional form for secretion of Hsp150. Hsp150 leaves the ER in the absence of retrograde COPI traffic, and the responsible determinant is a peptide repeated 11 times in the middle of the Hsp150 sequence. Herein, we localized the sorting determinant for Sec13p-independent ER exit to the C-terminal domain. Sec13p-dependent invertase left the ER in the absence of normal Sec13p function, when fused to the C-terminal domain of Hsp150, demonstrating that this domain contained an active mediator of Sec13p-independent secretion. Thus, Hsp150 harbors two different signatures that regulate its ER exit. Our data show that transport vesicles lacking functional Sec13p can carry out ER-to-Golgi transport, but select only specific cargo protein(s) for ER exit.

Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Delta erg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Delta erg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Delta erg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7-1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation. These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.

SummaryRecombinant DNA technology can be used to design and express collagen and gelatin-related proteins with predetermined composition and structure. Barley seed was chosen as a production host for a recombinant full-length collagen type I a1 (rCIa1) and a related 45-kDa rCIa1 fragment. The transgenic barley seeds were shown to accumulate both the rCIa1 and the 45-kDa rCIa1 fragment. Even when the amount of the rCIa1 was just above the detection threshold, this work using rCIa1 as a model demonstrated for the first time that barley seed can be used as a production system for collagen-related structural proteins. The 45-kDa rCI1a fragment expression, targeted to the endoplasmic reticulum, was controlled by three different promoters (a constitutive maize ubiquitin, seed endosperm-specific rice glutelin and germination-specific barley a-amylase fusion) to compare their effects on rCIa1 accumulation. Highest accumulation of the 45-kDa rCIa1 was obtained with the glutelin promoter (140 mg ⁄ kg seed), whereas the lowest accumulation was obtained with the a-amylase promoter. To induce homozygosity for stable 45-kDa rCIa1 production in the transgenic lines, doubled haploid (DH) progeny was generated through microspore culture. The 45-kDa rCIa1 expression levels achieved from the best DH lines were 13 mg ⁄ kg dry seeds under the ubiquitin promoter and 45 mg ⁄ kg dry seeds under the glutelin promoter. Mass spectroscopy and amino acid composition analysis of the purified 45-kDa rCIa1 fragment revealed that a small percent of prolines were hydroxylated with no additional detectable post-translational modifications.

The effect of abiotic stress responses on Potato virus A (PVA; genus Potyvirus) infection was studied. Salt, osmotic and wounding stress all increased PVA gene expression in infected Nicotiana benthamiana leaves. According to the literature, an early response to these stresses is an elevation in cytosolic Ca(2+) concentration. The infiltration of 0.1 m CaCl(2) into the infected leaf area enhanced the translation of PVA RNA, and this Ca(2+) -induced effect was more profound than that induced solely by osmotic stress. The inhibition of voltage-gated Ca(2+) channels within the plasma membrane abolished the Ca(2+) effect, suggesting that Ca(2+) had to be transported into the cytosol to affect viral gene expression. This was also supported by a reduced wounding effect in the presence of the Ca(2+) -chelating agent ethylene glycol tetraacetic acid (EGTA). In the absence of viral replication, the intense synthesis of viral proteins in response to Ca(2+) was transient. However, a Ca(2+) pulse administered at the onset of wild-type PVA infection enhanced the progress of infection within the locally infected leaf, and the virus appeared earlier in the systemic leaves than in the control plants. This suggests that the cellular environment was thoroughly modified by the Ca(2+) pulse to support viral infection. One message of this study is that the sensing of abiotic stress, which leads to cellular responses, probably via Ca(2+) signalling, associated with enhanced virus infection, may lead to higher field crop losses. Therefore, the effect of abiotic stress on plant viral infection warrants further analysis.

The endoplasmic reticulum (ER) is comprised of a controlled ratio of sheets and tubules, which are maintained by several proteins with multiple functions. Reticulons (RTNs), especially RTN4, and DP1/Yop1p family members are known to induce ER membrane curvature. RTN4B is the main RTN4 isoform expressed in non-neuronal cells. In this study, we identified FAM134C as a RTN4B interacting protein in mammalian, non-neuronal cells. FAM134C localized specifically to the ER tubules and sheet edges. Ultrastructural analysis revealed that overexpression of FAM134C induced formation of unbranched, long tubules or dense globular structures comprised of heavily branched narrow tubules. In both cases, tubules were non-motile. ER tubulation was dependent on the reticulon homology domain (RHD) close to the N-terminus. FAM134C plays a role in the autophagy pathway as its level elevated significantly upon amino acid starvation but not during ER stress. Moreover, FAM134C depletion reduced the number and size of autophagic structures and the amount of ER as a cargo within autophagic structures under starvation conditions. Dominant-negative expression of FAM134C forms with mutated RHD or LC3 interacting region (LIR) also led to the reduced number of autophagic structures. Our results suggest that FAM134C provides a link between regulation of ER architecture and ER turnover by promoting ER tubulation required for subsequent ER fragmentation and engulfment into autophagosomes. [Media: see text] [Media: see text] [Media: see text] [Media: see text]