Autophagy: many paths to the same end

Different mechanisms lead to the degradation of intracellular proteins in the lysosomal compartment. Activation of one autophagic pathway or another, under specific cellular conditions, plays an important role in the ability of the cell to adapt to environmental changes. Each form of autophagy has its own individual characteristics, but it also shares common steps and components with the others. This interdependence of the autophagic pathways confers to the lysosomal system, both specificity and flexibility on substrate degradation. We describe in this review some of the recent findings on the molecular basis and regulation for each of the different autophagic pathways. We also discuss the cellular consequences of their interdependent function. Malfunctioning of the autophagic systems has dramatic consequences, especially in non-dividing differentiated cells. Using the heart as an example of such cells, we analyze the relevance of autophagy in aging and cell death, as well as in different pathological conditions.     

Autophagy: Many paths to the same end

Different mechanisms lead to the degradation of intracellular proteins in the lysosomal compartment. Activation of one autophagic pathway or another, under specific cellular conditions, plays an important role in the ability of the cell to adapt to environmental changes. Each form of autophagy has its own individual characteristics, but it also shares common steps and components with the others. This interdependence of the autophagic pathways confers to the lysosomal system, both specificity and flexibility on substrate degradation. We describe in this review some of the recent findings on the molecular basis and regulation for each of the different autophagic pathways. We also discuss the cellular consequences of their interdependent function. Malfunctioning of the autophagic systems has dramatic consequences, especially in non-dividing differentiated cells. Using the heart as an example of such cells, we analyze the relevance of autophagy in aging and cell death, as well as in different pathological conditions. (Mol Cell Biochem 263: 55–72, 2004)     

N-linked glycosylation in Archaea: two paths to the same glycan

N‐linked protein glycosylation occurs in all three branches of life, eukaryotes, bacteria and archaea. The simplest system is that of the bacterium, Campylobacter jejuni, in which a heptasaccharide glycan is added to multiple proteins from a single lipid carrier molecule. In the eukaryotic system a conserved tetradecasaccharide modification is first added to target proteins, but is then modified by trimming and addition of other glycans from additional carrier molecules resulting in a diverse array of glycans of distinct functionality. In the halophilic Archaea from the Dead Sea, Haloferax volcanii, the surface array or S‐layer protein is glycosylated with a pentasaccharide. This glycan is synthesized from two separate carrier molecules, one that carries a tetrasaccharide and another that carries the terminal mannose, in a process that is analogous to that of eukaryotes. In this issue of Molecular Microbiology the glycosylation of the S‐layer of another halophilic Archaea from the Dead Sea, Haloarcula marismortui is characterized ( Calo et al., 2011 ). This S‐layer is glycosylated with the same pentasaccharide as that of Hfx. volcanii, but the intact pentasaccharide is synthesized on a single carrier molecule in Har. marismortui in a process that more closely resembles that of the bacterial N‐linked system.     

Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments

Polarized epithelial cells sort newly synthesized and recycling plasma membrane proteins into distinct trafficking pathways directed to either the apical or basolateral membrane domains. While the trans‐Golgi network is a well‐established site of protein sorting, increasing evidence indicates a key role for endosomes in the initial trafficking of newly synthesized proteins. Both basolateral and apical proteins have been shown to traverse endosomes en route to the plasma membrane. In particular, apical proteins traffic through either subapical early or recycling endosomes. Here we use the SNAP tag system to analyze the trafficking of the apical protein gp135, also known as podocalyxin. We show that newly synthesized gp135 traverses the apical recycling endosome, but not the apical early endosomes (AEEs). In contrast, post‐endocytic gp135 is delivered to the AEE before recycling back to the apical membrane. The pathways pursued by the newly synthesized and recycling gp135 populations do not detectably intersect, demonstrating that the biosynthetic and post‐endocytic pools of this protein are subjected to distinct sorting processes.      

The secretory apparatus of an ancient eukaryote: protein sorting to separate export pathways occurs before formation of transient Golgi-like compartments

Transmission of the protozoan parasite Giardia intestinalis to vertebrate hosts presupposes the encapsulation of trophozoites into an environmentally resistant and infectious cyst form. We have previously shown that cyst wall proteins were faithfully sorted to large encystation-specific vesicles (ESVs), despite the absence of a recognizable Golgi apparatus. Here, we demonstrate that sorting to a second constitutively active pathway transporting variant-specific surface proteins (VSPs) to the surface depended on the cytoplasmic VSP tail. Moreover, pulsed endoplasmic reticulum (ER) export of chimeric reporters containing functional signals for both pathways showed that protein sorting was done at or very soon after export from the ER. Correspondingly, we found that a limited number of novel transitional ER-like structures together with small transport intermediates were generated during encystation. Colocalization of transitional ER regions and early ESVs with coat protein (COP) II and of maturing ESVs with COPI and clathrin strongly suggested that ESVs form by fusion of ER-derived vesicles and subsequently undergo maturation by retrograde transport. Together, the data supported the hypothesis that in Giardia, a primordial secretory apparatus is in operation by which proteins are sorted in the early secretory pathway, and the developmentally induced ESVs carry out at least some Golgi functions.     

Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells

We have previously demonstrated that vacuolar sorting receptor (VSR) proteins are concentrated on prevacuolar compartments (PVCs) in plant cells. PVCs in tobacco (Nicotiana tabacum) BY-2 cells are multivesicular bodies (MVBs) as defined by VSR proteins and the BP-80 reporter, where the transmembrane domain (TMD) and cytoplasmic tail (CT) sequences of BP-80 are sufficient and specific for correct targeting of the reporter to PVCs. The genome of Arabidopsis (Arabidopsis thaliana) contains seven VSR proteins, but little is known about their individual subcellular localization and function. Here, we study the subcellular localization of the seven Arabidopsis VSR proteins (AtVSR1-7) based on the previously proven hypothesis that the TMD and CT sequences correctly target individual VSR to its final destination in transgenic tobacco BY-2 cells. Toward this goal, we have generated seven chimeric constructs containing signal peptide (sp) linked to green fluorescent protein (GFP) and TMD/CT sequences (sp-GFP-TMD/CT) of the seven individual AtVSR. Transgenic tobacco BY-2 cell lines expressing these seven sp-GFP-TMD-CT fusions all exhibited typical punctate signals colocalizing with VSR proteins by confocal immunofluorescence. In addition, wortmannin caused the GFP-marked prevacuolar organelles to form small vacuoles, and VSR antibodies labeled these enlarged MVBs in transgenic BY-2 cells. Wortmannin also caused VSR-marked PVCs to vacuolate in other cell types, including Arabidopsis, rice (Oryza sativa), pea (Pisum sativum), and mung bean (Vigna radiata). Therefore, the seven AtVSRs are localized to MVBs in tobacco BY-2 cells, and wortmannin-induced vacuolation of PVCs is a general response in plants.     

Vacuolar protein sorting: two different functional states of the AAA-ATPase Vps4p

The vacuolar protein sorting (Vps) pathway, in which Vps4 class I AAA-ATPases play a central role, regulates growth factor receptors, immune response, and developmental signaling, and participates in tumor suppression, apoptosis, and retrovirus budding. We present the first atomic structure of the nucleotide-free yeast His₆ΔNVps4p dimer and its AMPPNP (5′-adenylyl-β,γ-imidodiphosphate)-bound tetradecamer, derived from a cryo electron microscopy map. Vps4p dimers form two distinct heptameric rings and accommodate AAA cassettes in a head-to-head—not in a head-to-tail—fashion as in class II AAA-ATPases. Our model suggests a mechanism for disassembling ESCRT (endosomal sorting complex required for transport) complexes by movements of substrate-binding domains located at the periphery of the tetradecamer during ATP hydrolysis in one ring, followed by translocation through the central pore and ATP hydrolysis in the second ring.     

Meylin types with different protein components in the same species

Abstract— Myelin was isolated from bovine optic nerve, cerebral white matter, spinal cord white matter and peripheral nerve (intradural spinal roots). The freeze-dried myelin completely dissolved in phenol-formic acid-water (14:3:3, w/v/v), and acrylamide gel electrophoresis of the myelin proteins was performed with this solvent. Qualitative and quantitative differences were observed in the myelin proteins from the various regions of the CNS. Myelin of peripheral nerve contained proteins that are apparently unique to it and which are not found in the myelin of the CNS.     

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F₁ subunit of the ATP synthase (pF₁) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu²⁺ were inhibitory. Inhibition by Cu²⁺ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondria than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu²⁺ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF₁ with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu²⁺ were inhibitory. NEM, MPB, DTNB and Cu²⁺ inhibited import of the NEM-modified pF₁ into intact mitochondria. Import of pF₁ through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu²⁺ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.     

Subcellular compartments and protein topogenesis

A cell is surrounded by a plasma membrane. It contains various organelles, most of which are enclosed by limiting membranes. The intracellular space is thus divided into a number of subcellular compartments. Structurally, a cell is composed of membranes and the spaces enclosed by those membranes. In order to classify these compartments, the extracellular space has been designated S1 and whenever a unit membrane structure is crossed to arrive at the next space, one is added to term; the cytoplasmic space becomes S2, the intraluminal space of the endoplasmic reticulum and the intermembrane space of the mitochondria S3, and the matrix space of the mitochondria S4. Similarly, the plasma membrane is M1, the outer membrane of the mitochondria M2, and the inner counterpart M3. This classification of the subcellular compartments is useful in understanding a number of complicated cellular structures and functions. The intracellular transport of newly synthesized protein (protein topogenesis) and the probable development of subcellular organelles during phylogenesis of eukaryotic cells is discussed in terms of these subcellular compartments.     

TLR3 and TLR7 are targeted to the same intracellular compartments by distinct regulatory elements

Toll-like receptor (TLR) 3 and TLR7 are indispensable for host defense against viral infection by recognizing virus-derived RNAs and are localized to intracellular membranes via an unknown mechanism. We recently reported experiments with chimeric Toll-like receptors that suggested that the subcellular distribution of TLRs may be defined by their transmembrane and/or cytoplasmic domains. Here we demonstrate that the intracellular localization of TLR3 is achieved by a 23-amino acid sequence (Glu(727) to Asp(749)) present in the linker region between the transmembrane domain and Toll-interleukin 1 receptor resistance (TIR) domain. In contrast, the intracellular localization of TLR7 is achieved by its transmembrane domain. These elements also targeted a heterologous type I transmembrane protein CD25 to the intracellular compartment that contained TLR3 and TLR7. Despite their using distinct regulatory elements for intracellular localization, TLR3 was found to co-localize with TLR7. In addition, TLR3 and TLR7 were preferentially localized near phagosomes containing apoptotic cell particles. These findings reveal that TLR3 and TLR7 contain unique targeting sequences, which differentially lead them to the same intracellular compartments and adjacent to phagosomes containing apoptotic cell particles, where these receptors may access their ligands for the induction of immune responses against viral infection.