Lst1 and atg40 mutants have higher UPR response before and after DTT treatments, indicates that defeated in ER-phagy proteins will also induce the UPR.

ATZ-pYES2 is a vector natively expressing the ATZ, a misfolded protein, in yeast. ATZ-pYES2 used to observe a as substrate targeted for autophagy degradation.

Wild type, and mutants of the atg40 and lst1 were transformed with ATZ-pYES2 to observe whether loss-of-functions of atg40 and lst1 will lead to the accumulation of aggregated ATZ protein in the ER, which indicates that Atg40 and Lst1 are essential ER-phagy.

vacuolar protease-deficient yeast strain that lost protease activity wihle fail to degrade the autophagic cargo and served as negative control when compared with the wild-type original strain.

Atg8 protein is a marker protein to observe autophagosome formation. When Atg8 protein engineered with the red florescence protein (RFP), RFP-Atg8, RFP-Atg8 protein will be conjugated to the lipids and enable the membrane fusion to localize on the autophagosome.

In short, GFP-ATG8. GFP is a protein in the jellyfish Aequorea Victori that exhibits bright green fluorescence when excited at a wavelength of 488nm and has an emission peak at about 507nm ( blue to ultraviolet range).

GFP is served as biological marker for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression.

GFP consists of 238 amino acid with 27 kilo Dalton of the protein size. When ATG8 fused to GFP (GFP-ATG8), the ATG8 here as a protein of interest (ATG8 is a ubiquitin-like protein required for the formation of autophagosomal membranes) to carry the GFP which used as a reporter and exhibit green signal.

GFP-ATG8 will serve as a protein visualized marker gene which localized on the double membrane vesicle, the autophosome.

Only GST-Lst1 purified protein is able to bind with the Atg40 but not other subunits of COP11 coat proteins.

Atg8 also plays an important role in cargo recognition for selective autophagy by interacting with the receptor protein. When Atg40 is mutant, Lst1 is failed to interact and colocalize with Atg40, indicating that Lst1 is acting together with Atg40 to form autophagosome. And Sec23 is partially required with Atg40 to form autophagosome.

Bubble-shape like membrane bound transport vesicles that are made from the reticulum endoplasmic (ER) membrane, functions to carry the proteins (wanted or unwanted) and move from ER to Golgi apparatus before going to their destination, which is lysosome, peroxisomes or vacuole for degradation or recycle.

Here, refer to bubble-shaped membrane-bound transport vesicle that buddling from the endoplasmic reticulum (ER) membrane, functions to transport proteins from ER and fuse to golgi apparatus.

autophagy-deficient yeast strain which fail to form the autophagosomes to degrade and recycle substances.

SEC24C in mammalian cells is the homolog for Lst1 in yeast.

The mammals homolog of Atg40 in yeast. To verify the evolutionarily conserved function the ER-phagy receptor in higher order and complex organism such as in mammal cell, FAM134B, the homolog protein of Atg40 in mammals, was used to test its functional similarity.

To induce the UPR (unfolded protein response), DTT (dithiothreitol) is added to induce protein misfolding in the ER by blocking protein disulfide bond formation, thus activate and induce the UPR.

The relative intensity of GFP fluorescence signal in the cells was measured using the flow cytometry.

Torin 2 is a potent and selective mTOR inhibitor, which decreases cell viability and induces autophagy and apoptosis.

Lst1-Sec23 is a coat adaptor protein that carry the cargo molecules form ER to Golgi, which mainly to help the secretory proteins transportation.

Protein recycling and self-eating process that selectively happens in endoplasmic reticulum terms as ER-phagy (phagy means eating). Selective autophagy requires a receptor protein to recognize its degradation targets and directs the targets to be sequestrated by autophagosomes, a double membrane vesicle structure, which to be transported into lysosomes or vacuoles.

A protein named as Atg40, has been recognized as a ER-phagy receptor protein in yeast. FAM134B is a putative mammals homologue of ATG40.

Literature first explain the Atg40 ER-phagy receptor https://doi.org/10.1038/nature14506 (ATG40)

https://doi.org/10.1038/nature14498 (FAM134B)

Lst1-Sec23 is a coat adaptor protein that carry the cargo molecules form ER to Golgi, which mainly to help the secretory proteins transportation. The author discovered new function of the Lst1/Sec23 in self-eating and protein recycling process (autophagy), in which the Lst1-Sec23 functions together with the autophagy receptor, Atg40, to localize the ER domains for autophagic degradation.

ULK1 is one of two mammalian homologues of the yeast ATG1 kinase, known for its role in autophagy initiation

A semi-quantitative GFP-Atg8 cleavage assay to observe the vacuolar delivery of GFP-Atg8 by bulk autophagy assessed by Western blotting using an anti-GFP antibody.

To measure the bulk autophagic flux from the cytosol into the vacuole, the Pho8Δ60 assay measures the enzymatic activity resulting from delivery to the vacuole from cytosol. The genetic engineered Pho8 truncated of 60 amino acid of the N-terminal fragment will remain in the cytosol and is delivered to the vacuole only through bulk autophagy.

A journal article to explain assays to monitor autophagy in yeast. https://doi.org/10.3390/cells6030023

Sec61 as ER translocon protein marker and FM4-64 as vacuolar membrane marker. From the Fig 1 A, the overlapping green and red signals in the vacuolar membrane were decreased in the mutants lst1 and atg40 compared to WT, sec24-3A iss1 mutant and iss1 mutant, which were quantified in Fig 1 B, indicated that the Lst1 and ATG40 are required for degradation of ER in the vacuole .

Two other yeast SEC24 paralogs, lst1 and issI, are also cargo protein of COPII complexes. Mammalian cells have four SEC24 homologues.

The loss of function of the SEC24 paralogs in yeast, lst1Δ, iss1Δ, were used to determine which subunit of the SEC24 coat protein is involved specifically in the selective ER-phagy, but not the non-selective bulk autophagy.

sec24-3A mutant is the phosphorylation site mutated mutant from changed from Threonine to Alanine amino acid (Sec24-T324A/T325A/T328A = Sec24-3A mutant).

Rapamycin is a drug to induce ER stress by inhibiting the mTOR pathway in the autophagy process. Long incubation time is refer to 24hrs.

Key question raised by the authors, how the dispersed site throughout the ER are recognized by the selective autophagy receptors to deliver the cargo for degradation.

The authors hypothesized that, COP11 coat proteins which involved in membrane-budding at the ER, could involve in ER-phagy.

First ER-phagy receptors that were identified in yeast is ATG40, and FAM134B in mammals.

What are the differences between proteasome and autophagosome?

No additive effect was observed when double mutant of lst1atg40 is investigated. This finding drawn another summary which LST1 and ATG40 function in the same pathway.

To investigate how the role of ER-phagy receptor, ATG40, involved in packaging the cargo for recycling in ER specifically, the authors made the connection of observation and results obtained in ATG40, and associated with LST1 to hypothesize that LST1 could be associated with the ER-phagy receptor, ATG40.

To examine the association of two proteins in the cell, colocalization of two fusion proteins with different florescence tagged fused with two proteins are utilized to be visualized under the confocal/florescence microscope, to test whether these two signals (red and green florescence in this study) could be overlapping in the cell.

LST1 fused with GFP, the green florescence protein. ATG40 fused with mCherry, the red florescence protein.

In yeast, the ER consists of 3 subdomains, the cytoplasmic ER, the cortical ER, and the perinuclear ER (also called nuclear envelope).

Atg39 specifically localizes to the nuclear envelope. Therefore, the Atg39-dependent pathway in selective autophagy terms as nucleophagy.

Conversely, Atg40 predominantly localizes to the cytoplasmic ER and the cortical ER, and loads fragments (ER tubules or sheets) of these ER subdomains into the phagophore. Thus, Atg40 is exclusively involved in ER-phagy.

To exclude the possibilities that LST1 is localized to the nuclear envelope and do not involve in nucleophagy, the authors examine the colocalization of Atg39 and Lst1, and rule out the possibility that the Lst1 is localized in nuclear envelope and do not reside on the nuclear envelope and its marker, Hmg1.

A conclusion summarized by the authors based on experiments that have been done to distinguish the bulk autophagy and selective autophagy that happened in various organelles, and only ER-phagy is required by Lst1 coat protein.

To draw a conclusion which exclusively functional in one particular experiment, the designs of various experiments are needed in order to rule out other possibilities.

Lnp1 is a protein to maintain the structure of ER by stabilizing rearrangements of the ER networks.

Further reading: https://doi.org/10.1073/pnas.1805032115

Only GST-Lst1 purified protein is able to bind with the Atg40 but not other subunits of COP11 coat proteins.

This experiment is an in vitro binding assay by purifying the GST fusion of target coat proteins of the COPII and incubated with Atg40 fusion protein with FLAG epitope tagged expressed in the yeast.

Bulk autophagy also called non-selective macroautophagy. During bulk autophagy, autophagosomes fuse with lysosomes or vacuoles resulting in the degradation of the cargo.

While during selective autophagy, the cargo is distinguished (mitochondria, ER, ribosomes, peroxisomes and etc.) and recognized by specific cargo receptors, or selective autophagy receptors, to connect with the autophagosome for degradation.

https://www.youtube.com/watch?v=Gc9gx33GvF0&t=2s https://www.youtube.com/watch?v=Ws0mOmfC9EU

The authors tested whether or not the defeated of Lst1 in yeast will have the similar observation in mammalian cells by looking at the homologs of the isoforms of Sec24 in mammals.

Pep4, a vacuolar protease required for function of multiple hydrolases, is required for autophagic cargo degradation

The ER exit sites are specialized ER zones for the transport of cargo proteins from the ER to the Golgi apparatus.

The degradation of damaged mitochondria in the cell through selective autophagy.

A type of selective autophagy to degrade a specific organelle called peroxisomes in the cell.

Nuclear component that is selectively degraded via autophagy pathway.

Nucleophagy and ER-phagy

Misfolded protein are retained in the ER and subjected for proteosomal degradation which occurs in the cytosol.

Learning from the youtube:

https://www.youtube.com/watch?v=fEEbyYjXNUU

When newly synthesized protein is not properly folded into their correct orientation in the endoplasmic reticulum (ER), the ER now is under the stress to degrade the misfolded protein. To degrade the misfolded proteins and maintain the ER homeostasis, the cellular signaling will turn on the unfolded protein response to help the cell deals with problematic proteins for recycle and degradation.

https://www.youtube.com/watch?v=vy4m-fUOn9o

Genes are commonly generate isoforms from the same locus to produce different mRNA and thus give rise to different length of amino acid with similar protein functions.

Lst1 and SEC24C, are homologous gene in yeast and mammalian respectively. The protein or DNA sequence of these genes, Lst1 and SEC24C, are highly similar and evolutionary share the common ancestor, termed as homolog.

ER is a transportation system of the eukaryotic cell composed of network of tubules and sheet-like area adjacent with the nuclear envelope and stretches out to the cell periphery.

Learning corner with video: https://www.youtube.com/watch?v=an7tpWR16mo