Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

Background： The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1,6- and 26-d-old pigs were studied during 1） euinsulinemic-euglycemic-euaminoacidemic, 2） euinsulinemic-euglycemiohyperaminoacidemic, and 3） hyperinsulinemic-euglycemic-euaminoacidemic clamps for 2 h. In experiment 2, 5-d-old pigs were studied during 1） euinsulinemic-euglycemic-euaminoacidemic-euleucinemic, 2） euinsulinemic-euglycemic-hypoaminoacidemic- hyperleucinemic, and 3） euinsulinemic-euglycemic-euaminoacidemic-hyperleucinemic clamps for 24 h. We determined in muscle indices of ubiquitin-proteasome, i.e., atrogin-1 （MAFbx） and muscle RING-finger protein-1 （MuRF1） and autophagy-lysosome systems, i.e., unc51-1ike kinase 1 （UKL1）, microtubule-associated protein light chain 3 （LC3）, and lysosomal-associated membrane protein 2 （Lamp-2）. For comparison, we measured ribosomal protein 56 （rpS6） and eukaryotic initiation factor 4E （elF4E） activation, components of translation initiation. Results： Abundance of atrogin-1, but not MuRF1, was greater in 26- than 6-d-old pigs and was not affected by insulin, amino acids, or leucine. Abundance of ULK1 and LC3 was higher in younger pigs and not affected by treatment. The LC3-11/LC3-1 ratio was reduced and ULK1 phosphorylation increased by insulin, amino acids, and leucine. These responses were more profound in younger pigs. Abundance of Lamp-2 was not affected by treatment or development. Abundance of elF4E, but not rpS6, was higher in 6- than 26-d-old-pigs but unaffected by treatment. Phosphorylation of elF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the responses decreased with development.

Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

Bacillus thuringiensis(Bt)cotton production is challenged by two main problems,i.e.,the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs.Therefore,increasing the Bt protein concentration at the boll stage,especially in bolls,has become the main goal for increasing insect resistance in cotton.In this study,two protein degradation inhibitors(ethylene diamine tetra acetic acid(EDTA)and leupeptin)were sprayed on the bolls,subtending leaves,and whole cotton plants at the peak flowering stage of two Bt cultivars(medium maturation Sikang 1(SK1))and early maturation Zhongmian 425(ZM425)in 2019 and 2020.The Bt protein content and protein degradation metabolism were assessed.The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8%and 25.0 to 38.6%in the treated bolls of SK1 and ZM425 respectively,while they were decreased in the subtending leaves of these treated bolls.In the treated leaves,the Bt protein concentrations increased by 7.6 to 23.5%and 11.2 to 14.9%in SK1 and ZM425,respectively.The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves.The Bt protein concentrations in bolls were higher,increasing by 22.5 to 31.0%and 19.6 to 32.5%for SK1 and ZM425,respectively.The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents,protease and peptidase activities and significant enhancements in soluble protein contents.These results indicated that inhibiting protein degradation could improve the protein content,thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants.Therefore,the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

ATP regulates the phosphorylation and degradation of myofibrillar proteins in ground ovine muscle

Phosphorylation post-translational modification plays an important role in postmortem muscle quality traits. Adenosine triphosphate(ATP) is an energy source and a key substrate of phosphorylation which provides the phosphatase groups to proteins in the presence of protein kinases. However, in postmortem muscle, the effects of ATP content on phosphorylation are poorly studied. The study investigated the effect of ATP on protein phosphorylation and degradation in postmortem ovine muscle. The ground muscle with/without additional ATP were treated/control groups and stored at 25 and 4℃, respectively. The ATP content led to different changes of p H value between the ATP-treated and control groups. The phosphorylation level of myofibrillar proteins was higher(P<0.05) in ATP-treated group compared to the control group at both temperatures, which suggested that ATP played a vital role in postmortem protein phosphorylation. A slower degradation rate of μ-calpain, desmin and troponin T was observed in the ATP-treated group which showed that there was a negative relationship between ATP level and the degradation of proteins. These observations clearly highlighted the role of ATP on the development of meat quality by regulating the phosphorylation and degradation of myofibrillar proteins in postmortem ovine muscle.

Non-small molecule PROTACs(NSM-PROTACs):Protein degradation kaleidoscope

The proteolysis targeting chimeras(PROTACs)technology has been rapidly developed since its birth in 2001,attracting rapidly growing attention of scientific institutes and pharmaceutical companies.At present,a variety of small molecule PROTACs have entered the clinical trial.However,as small molecule PROTACs flourish,non-small molecule PROTACs(NSM-PROTACs)such as peptide PROTACs,nucleic acid PROTACs and antibody PROTACs have also advanced considerably over recent years,exhibiting the unique characters beyond the small molecule PROTACs.Here,we briefly introduce the types of NSM-PROTACs,describe the advantages of NSM-PROTACs,and summarize the development of NSM-PROTACs so far in detail.We hope this article could not only provide useful insights into NSM-PROTACs,but also expand the research interest of NSM-PROTACs.

Enzyme-Catalyzed Activation of Pro-PROTAC for Cell-Selective Protein Degradation

Proteolysis targeting chimera(PROTAC)technology is a chemical protein knockdown approach that degrades protein by hijacking the cellular ubiquitinproteasome system to impede tumor growth.Its therapeutic potential,however,isdifficult to define due to the lack of control over the cell selectivity of PROTACs,in particular,if the therapeutic purpose is to be executedin a specific cell type.Herein,we report the design of a Pro-PROTAC and its catalytic activation of the endogenous overexpressed enzyme in cancer cells for cellselective protein degradation.We demonstrate that the chemical modification of the binding site between PROTAC and E3 ligase with trimethyl-locked quinone efficiently blocks the protein degradation capability of PROTAC.However,NAD(P)H quinone dehydrogenase 1(NQO1),an enzyme overexpressed in cancer cells,could reduce the trimethyl-locked quinone to remove the chemical modification and activate NQO1-PROTAC for cancer cell-selective protein degradation.Further,we show that NQO1-catalyzedβ-Lapachone reduction potentiated cellular oxidative stress to activate aryl boronic acid-caged ROS-PROTAC in living cells for bromodomain-containing protein 4 degradation with enhanced cell selectivity.Collectively,our strategy of designing Pro-PROTAC in response to endogenous species of diseased cells expands the chemical biology toolbox for cell-selective protein degradation and would be of great interest in targeted therapeutics discovery.

Nucleolus-localized Def-CAPN3 protein degradation pathway and its role in cell cycle control and ribosome biogenesis

The nucleolus,as the‘nucleus of the nucleus’,is a prominent subcellular organelle in a eukaryocyte.The nucleolus serves as the centre for ribosome biogenesis,as well as an important site for cell-cycle regulation,cellular senescence,and stress response.The protein composition of the nucleolus changes dynamically through protein turnover to meet the needs of cellular activities or stress responses.Recent studies have identified a nucleolus-localized protein degradation pathway in zebrafish and humans,namely the Def-CAPN3 pathway,which is essential to ribosome production and cell-cycle progression,by controlling the turnover of multiple substrates(e.g.,ribosomal small-subunit[SSU]processome component Mpp10,transcription factor p53,check-point proteins Chk1 and Wee1).This pathway relies on the Ca2þ-dependent cysteine proteinase CAPN3 and is independent of the ubiquitin-mediated proteasome pathway.CAPN3 is recruited by nucleolar protein Def from cytoplasm to nucleolus,where it proteolyzes its substrates which harbor a CAPN3 recognition-motif.Def depletion leads to the exclusion of CAPN3 and accumulation of p53,Wee1,Chk1,and Mpp10 in the nucleolus that result in cell-cycle arrest and rRNA processing abnormality.Here,we summarize the discovery of the Def-CAPN3 pathway and propose its biological role in cell-cycle control and ribosome biogenesis.

Machine Learning Modeling of Protein-intrinsic Features Predicts Tractability of Targeted Protein Degradation

Targeted protein degradation(TPD)has rapidly emerged as a therapeutic modality to eliminate previously undruggable proteins by repurposing the cell’s endogenous protein degradation machinery.However,the susceptibility of proteins for targeting by TPD approaches,termed“degradability”,is largely unknown.Here,we developed a machine learning model,model-free analysis of protein degradability(MAPD),to predict degradability from features intrinsic to protein targets.MAPD shows accurate performance in predicting kinases that are degradable by TPD compounds[with an area under the precision–recall curve(AUPRC)of 0.759 and an area under the receiver operating characteristic curve(AUROC)of 0.775]and is likely generalizable to independent non-kinase proteins.We found five features with statistical significance to achieve optimal prediction,with ubiquitination potential being the most predictive.By structural modeling,we found that E2-accessible ubiquitination sites,but not lysine residues in general,are particularly associated with kinase degradability.Finally,we extended MAPD predictions to the entire proteome to find964 disease-causing proteins(including proteins encoded by 278 cancer genes)that may be tractable to TPD drug development.

Spatiotemporal regulation of ubiquitin-mediated protein degradation via upconversion optogenetic nanosystem

Protein degradation technology,which is one of the most direct and effective ways to regulate the life activities of cells,is expected to be applied to the treatment of various diseases.However,current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation,making them difficult to transform into clinical applications.In this article,an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation.This system worked via two interconnected parts:1)the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure;2)the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation.Experimental results based on model protein(Green Fluorescent Protein,GFP)validated that this system could fulfill protein degradation both in vitro(both Hela and 293T cells)and in vivo(by upconversion optogenetic nanosystem),and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time(0–25 h)and the illumination frequency(the illuminating frequency of 0–30 s every 1 min).We further took another functional protein(The Nonstructural Protein 9,NSP9)into experiment.Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus(PRRSV)was inhibited by degrading the NSP9 in this light-induced system,and PRRSV proliferation was affected by different light conditions(illumination time varies from 0–24 h).We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.

The role of 5′-adenosine monophosphate-activated protein kinase(AMPK)in skeletal muscle atrophy

As a key coordinator of metabolism,AMP-activated protein kinase(AMPK)is vitally involved in skeletal muscle maintenance.AMPK exerts its cellular effects through its function as a serine/threonine protein kinase by regulating many downstream targets and plays important roles in the development and growth of skeletal muscle.AMPK is activated by phosphorylation and exerts its function as a kinase in many processes,including synthesis and degradation of proteins,mitochondrial biogenesis,glucose uptake,and fatty acid and cholesterol metabolism.Skeletal muscle atrophy is a result of various diseases or disorders and is characterized by a decrease in muscle mass.The pathogenesis and therapeutic strategies of skeletal muscle atrophy are still under investigation.In this review,we discuss the role of AMPK in skeletal muscle metabolism and atrophy.We also discuss targeting AMPK for skeletal muscle treatment,including exercise,AMPK activators including 5-amino-4-imidazolecarboxamide ribonucleoside and metformin,and low-level lasers.These studies show the important roles of AMPK in regulating muscle metabolism and function;thus,the treatment of skeletal muscle atrophy needs to take into account the roles of AMPK.

2007 International Symposium on Protein Modification and Degradation in Beijing(SPMDB2007)

Presidents of Symposium Depei Liu,President of Chinese Academy of Medical Sciences Zhu Chen,Vice President of Chinese Academy ofSciences.

Design and Synthesis of Novel Bispecific Molecules for Inducing BRD4 Protein Degradation

Proteolysis targeting chimeras（PROTACs） are bispecific molecules containing a target protein binder and a ubiquitin ligase binder connected by a linker. Recently, some heterobifunctional small molecule bromodomain-containing protein 4（BRD4） degraders based on the concept of PROTACs were designed to induce the degradation of BRD4 protein. Herein, we synthesized a new class of PROTAC BRD4 degraders. One of the most promising compound 22f exhibited robust potency of BRD4 inhibition with IC50 value of （9.4±0.6） nmol/L. Furthermore, com- pound 22f potently inhibited cell proliferation in BRD4-sensitive cell lines RS4;11 with IC50 value of （27.6±1.6） nmol/L and capable of inducing degradation of BRD4 protein at 0.5-1.0 μmol/L in the RS4;11 cells. These data establish that compound 22f is a potent and efficacious BRD4 degrader.

Degradation of Cry1Ab Protein Within Transgenic Bt Maize Tissue by Composite Microbial System of MC1

Environmental safety issues involved in transgenic plants have become the concern of researchers, practitioners and policy makers in recent years. Potential differences between Bt maize（ND1324 and ND2353 expressing the insecticidal Cry1Ab protein） and near-isogenic non-Bt varieties（ND1392 and ND223） in their influence on the composite microbial system of MC1 during the fermentation process were studied during 2011-2012. Cry1Ab protein in Bt maize residues didn＇t affect characteristics of lignocellulose degradation by MC1, pH of fermentation broth decreasing at initial stage and increasing at later stage of degradation. The quality of various volatile products in fermentation broth showed that no signifi cant difference of residues fermentation existed between Bt maize and non-Bt maize. During the fermentation MC1 efficiently degraded maize residues by 83%-88%, and cellulose, hemicelluloses and lignin content decreased by 70%-72%, 72%-75% and 30%-37%, respectively. Besides that, no consistent difference was found between Bt and non-Bt maize residues lignocellulose degradation by MC1 during the fermentation process. MC1 degraded 88%-89% Cry1Ab protein in Bt maize residues, and in the fermentation broth of MC1 and bacteria of MC1 Cry1Ab protein was not detected. DGGE profi le analyses revealed that the microbial community drastically changed during 1-3 days and became stable until the 9th day. Though the dominant strains at different fermentation stages had signifi cantly changed, no difference on the dominant strains was observed between Bt and non-Bt maize at different stages. Our study indicated that Cry1Ab protein did not infl uence the growth characteristic of MC1.

The role of sequestosome 1/p62 protein in amyotrophic lateral sclerosis and frontotemporal dementia pathogenesis

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are multifaceted diseases with genotypic,pathological and clinical overlap.One such overlap is the presence of SQSTM1/p62 mutations.While traditionally mutations manifesting in the ubiquitin-associated domain of p62 were associated with Paget’s disease of bone,mutations affecting all functional domains of p62 have now been identified in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.p62 is a multifunctional protein that facilitates protein degradation through autophagy and the ubiquitin-proteasome system,and also regulates cell survival via the Nrf2 antioxidant response pathway,the nuclear factor-kappa B signaling pathway and apoptosis.Dysfunction in these signaling and protein degradation pathways have been observed in amyotrophic lateral sclerosis and frontotemporal lobar degeneration,and mutations that affect the role of p62 in these pathways may contribute to disease pathogenesis.In this review we discuss the role of p62 in these pathways,the effects of p62 mutations and the effect of mutations in the p62 modulator TANK-binding kinase 1,in relation to amyotrophic lateral sclerosis-frontotemporal lobar degeneration pathogenesis.

Isolation,Identification of Bacillus Thuringiensis/Cereus and Its Enhancement on Protein Wastewater Treatment by Rhodobacter Sphaeroides

In order to enhance the degrading protein capability of purple non-sulfur bacteria(PNSB),an effective strain,L2,was used to co-culture with Rhodobacter sphaeroides ATCC17023.The effects of added strain on protein removal of R.sphaeroides were investigated.Results showed that strain L2,being identified as Bacillus thuringiensis/cereus,had a high potential for producing protease with a production of 295 U/m L.The optimal B.thuringiensis/cereus(40 μL) could significantly increase protein degradation of R.sphaeroides.Protein removal and biomass production were improved by 483% and 67%,respectively.R.sphaeroides/total biomass production was more than 95%.Theoretical analysis revealed that R.sphaeroides syntrophically interacted with B.thuringiensis/cereus.Protein degradation of B.thuringiensis/cereus provided small molecule substrates(VFAs) for R.sphaeroides growth and cells materials synthesis.

Issues with Tropical and Temperate Ensilage Protein and Amino Acid Feeds Utilization: A Research Note

Alfalfa protein breakdown was to soluble NPN of oligopeptide-N, AA-N, amide-N, amine-N and NH3-N. Acidity (pH) and moisture (Aw) are critical in determining extent of fermentation and changes in composition. Further changes in digestive flows and post-prandial plasma AA are indicators of protein status. Dual-purpose cropping and tree plant cropping was with ensiling management of the undergrowth. On-farm field-drying and probiotic additives are promising. It is suggested acidity with propionic acid and microbial inoculants together with field-drying and chop length are required to optimize profile qualities in silage. It is proposed use of denaturing with acid and dust cropping with a hypothetical PNA-Auxin repressor to plant protease. Further study with field-drying to follow is needed. Feeding HIS, ARG and LEU AA supplement to change GRH and GH profiles could be used to promote LBM in production. Dual-purpose cropping can expand subsistence to mixed farming with expanded livestock products and services and resources. PNA-Auxin and PNA-ARF penetrates the plant shoot tips to deliver a TF mRNA to boost proteins in residual cell tissues. Ensiled % AA-N delivery per os to per duodenum was higher;yet total AA-N flow was higher in the control. It is suggested that “bulk” flow was less but with a “tighter” conversion on TAA. FAA was 145% higher in the ensiled versus the fresh control indicating the ENU with less PFAA supplied. FAA on the ensiled diet is high inferred to be more soluble and escape lower from the rumen. WSC are less supplied in fermented forage with VFA being lower and presenting the question whether WSC should be supplied for energy and also with EFE through breaking down of polymers of lignocellulose. It was surmised, although not known, that higher dilution rate (% hr-1) was true on the fresh diet compared to the ensiled although end-products may initially detract with feed but that further digestion in the fresh feed may be higher with intake. Plasma AA before and after absorption or feeding are indicators of synthesis and breakdown. No data was available on N status;protein nutrition on neat silage was probably due to net efflux of AA with mobilization before influx with feeding and subsequent insulin action for uptake. Estuarine aquatic plant spp., water hyacinth used in the Philippines and duckweed studies in Australia, and post-harvest treatment with chemical additives and anti-microbial agents to help control potential transfer of diseases. “Greens” as supplements has yet to be established for anti-microbial properties for animal health and welfare. In conclusion, alfalfa silage fed at standard 0.6 cm particle size and wilted led to dramatic changes with AA breakdown, dramatic changes in duodenal AA flows from escape and recapture into microbial cells. Also N status of animals was compromised by lack of adequate “stores”, mobilized, resulting in a net decrease in total plasma AA with insulin-dependent uptake to tissue.

Winery by‑products as a feed source with functional properties:dose–response effect of grape pomace,grape seed meal,and grape seed extract on rumen microbial community and their fermentation activity in RUSITEC

Background Grape and winery by-products have nutritional values for cattle and also contain functional compounds like phenols,which not only bind to protein but can also directly affect microbiota and their function in the rumen.We characterized the nutritional and functional effects of grape seed meal and grape pomace as well as an effective dosage of grape phenols on ruminal microbiota and fermentation characteristics using a rumen simulation technique.Results Six diets(each n=8)were compared including a control diet(CON,no by-product),a positive control diet(EXT,CON+3.7%grape seed extract on a dry matter(DM)basis),two diets with grape seed meal at 5%(GS-low)and 10%(GS-high),and two diets with grape pomace:at 10%(GP-low)and 20%(GP-high),on a DM basis.The inclusion of the by-product supplied total phenols at 3.4%,0.7%,1.4%,1.3%,and 2.7%of diet DM for EXT,GS-low,GS-high,GP-low,and GP-high,respectively.Diets were tested in four experimental runs.All treatments decreased ammonia concentrations and the disappearances of DM and OM(P<0.05)compared to CON.EXT and GP-high lowered butyrate and odd-and branch-chain short-chain fatty acids while increased acetate compared to CON(P<0.05).Treatments did not affect methane formation.EXT decreased the abundance of many bacterial genera including those belonging to the core microbiota.GP-high and EXT consistently decreased Olsenella and Anaerotipes while increased Ruminobacter abundances.Conclusion The data suggest that the inclusion of winery by-products or grape seed extract could be an option for reducing excessive ammonia production.Exposure to grape phenols at a high dosage in an extract form can alter the rumen microbial community.This,however,does not necessarily alter the effect of grape phenols on the microbial community function compared to feeding high levels of winery by-products.This suggests the dominant role of dosage over the form or source of the grape phenols in affecting ruminal microbial activity.In conclusion,supplementing grape phenols at about 3%of diet DM is an effective dosage tolerable to ruminal microbiota.

Targeted Degradation of DNA/RNA Binding Proteins via Covalent Hydrophobic Tagging

Targeted protein degradation(TPD)holds great promise for biological inquiry and therapeutic development.However,it still remains elusive to destruct DNA/RNA binding proteins(DBPs/RBPs)previously deemed undruggable.Herein,we report ligandassisted covalent hydrophobic tagging(LACHT)as a modular strategy for TPD of these difficult-totarget proteins.Guided by a noncovalent protein ligand,LACHT leverages a reactive N-acyl-N-alkyl sulfonamide group to covalently label the protein target with a hydrophobic adamantane,which further engages the cellular quality control mechanism to induce proteolytic degradation.Using a smallmolecule ligand,we demonstrated that LACHT allowed TPD of a DBP,bromodomain-containing protein 4,in human leukemia cells with high efficiency.Mechanistic studies revealed that LACHT-mediated TPD dependent on ligand-directed irreversible tagging and the covalently labeled proteins underwent polyubiquitination before removal through both the proteasome and the lysosome.Furthermore,when an RNA ligand was employed,we showed that LACHT also enabled TPD of an RBP,Lin28a,leading to upregulation of its downstream let-7 miRNA.This study thus provides a generalizable platform to expand the TPD toolbox for biomedical applications.

Artificial Nucleic Acid Tractor-Directed Simultaneous Depletion of Oncogenic Membrane Proteins Without Hijacking Proteolysis-Specific Actuator

Targeted protein degradation(TPD)is an emerging tool for degrading proteins of interest,which affords an attractive modality for cancer therapy.However,the present TPD technologies must engage a proteolysis-specific actuator to initiate degradation of targeted proteins in the proteasome or lysosome.Herein,we report an artificial tractor that can induce endocytosis-mediated protein depletion without hijacking a proteolysis-specific actuator.In this design,bispecific aptamer chimeras(BSACs)are established,which can bridge human epidermal growth factor receptor 2(ErbB-2),an important biomarker in a common important biomarker in cancer,with membrane proteins of interest.Taking advantage of the property of aptamer-induced endocytosis and digestion of ErbB-2,another membrane protein is translocated into the lysosome in a hitchhike-like manner,resulting in lysosomal proteolysis along with ErbB-2.This strategy frees the TPD from the fundamental limitation of proteolysis-specific actuator and allows simultaneous regulation of the quantity and function of two oncogenic receptors in a cell-type-specific manner,expanding the application scope of TPD-based therapeutics.

Plasma Membrane Protein Ubiquitylation and Degradation as Determinants of Positional Growth in Plants

Being sessile organisms, plants evolved an unparalleled plasticity in their post-embryonic development, allowing them to adapt and fine-tune their vital parameters to an ever-changing environment. Cross-talk between plants and their environment requires tight regulation of information exchange at the plasma membrane （PM）. Plasma membrane proteins mediate such communication, by sensing variations in nutrient availability, external cues as well as by controlled solute transport across the membrane border. Localiza-tion and steady-state levels are essential for PM protein function and ongoing research identified cis- and trans-acting determinants, involved in control of plant PM protein localization and turnover. In this overview, we summarize recent progress in our understanding of plant PM protein sorting and degradation via ubiquitylation, a post-translational and reversible modification of proteins. We highlight characterized components of the machinery involved in sorting of ubiquitylated PM proteins and discuss consequences of protein ubiquitylation on fate of selected PM proteins. Specifically, we focus on the role of ubiquitylation and PM protein degradation in the regulation of polar auxin transport （PAT）. We combine this regulatory circuit with further aspects of PM protein sorting control, to address the interplay of events that might control PAT and polarized growth in higher plants.

Protein Domains Involved in Assembly in the Endoplasmic Reticulum Promote Vacuolar Delivery when Fused to Secretory GFP, Indicating a Protein Quality Control Pathway for Degradation in the Plant Vacuole

The correct folding and assembly of newly synthesized secretory proteins are monitored by the protein quality control system of the endoplasmic reticulum （ER）. Through interactions with chaperones such as the binding protein （BiP） and other folding helpers, quality control favors productive folding and sorts for degradation defective proteins. A major route for quality control degradation identified in yeast, plants, and animals is constituted by retrotranslocation from the ER to the cytosol and subsequent disposal by the ubiquitin/proteasome system, but alternative routes involving the vacuole have been identified in yeast. In this study, we have studied the destiny of sGFP418, a fusion between a secretory form of GFP and a domain of the vacuolar protein phaseolin that is involved in the correct assembly of phaseolin and in BiP recognition of unassembled subunits. We show that sGFP418, despite lacking the phaseolin vacuolar sorting signal, is delivered to the vacuole and fragmented, in a process that is inhibited by the secretory traffic inhibitor brefeldin A. Moreover, a fusion between GFP and a domain of the maize storage protein γ-zein involved in zein polymerization also undergoes post-translational fragmentation similar to that of sGFP418. These results show that defective secretory proteins with permanently exposed sequences normally involved in oligomerization can be delivered to the vacuole by secretory traffic. This strongly suggests the existence of a plant vacuolar sorting mechanism devoted to the disposal of defective secretory proteins.