Intracellular Compartments and Protein Sorting September 3, 2019 Tom Gallagher tgallag@luc.edu CTRE, Room 234 extension 64850

Intracellular Compartments See also “Visual Guide to Human Cells” www.allencell.org/visual-guide-to-human-cells

A map of protein trafficking in a eukaryotic cell

Sorting signals direct proteins to specific compartments

Some Typical Signal Peptides Function of Signal Peptide Example of Signal Peptide Import into ER Retain in lumen of ER Import into mitochondrial matrix Import into nucleus Export from nucleus Import into peroxisomal matrix Usually at the N-terminus; about 30-50 residues in length, central region is often hydrophobic Usually at the C-terminus, short 4-residue KDEL is a common ER retention signal Usually at the N-terminus, about 30 residues in length, often an amphipathic alpha helix Usually NOT at protein termini; a positive charged sequence is common, i.e., KKKRK Diverse, but always distinctly different than nuclear import signal, i.e., HLLALKLAGLDI Usually at the C-terminus, i.e., SKL-COOH

Gated Transport between Cytosol and Nucleus

The nucleus is bounded by a double membrane that is perforated with nuclear pores

The nuclear pore is type of molecular sieve

Ran proteins control nuclear import and export

Ran proteins control nuclear import and export

Nuclear import and export: IMPORTANCE In cancers: Several human cancers overexpress export receptors; dysregulating import/export In biological therapeutics: Several experimental therapeutics; i.e., CRISPR/CAS; operate in nuclei (need to know about nuclear import and export) In cell signaling: Several signaling systems rely on nuclear import for temporal and spatial control

Cell signaling : Control at nuclear import stage

Transmembrane Transport between Cytosol and Mitochondria

The mitochondrion is bounded by two membranes, creating four compartments

The mitochondrial matrix import signal peptide and its interaction with the mitochondrial import receptor

Protein import into the mitochondrial matrix requires the TOM and TIM complexes

Protein import into mitochondria requires chaperones and an electrochemical gradient

Mitochondria : IMPORTANCE Mitochondrial disease: Collection of very rare genetic diseases causing poor growth, developmental delays, muscle weakness Mutations in the mitochondrial DNA genome are largely responsible; however it is notable that mutations in either mtDNA, or the nuclear DNA genes encoding mt proteins, can cause similar disease symptoms (a case of phenocopying here) Impaired import of mitochondrial proteins: One clinical example in Huntington’s Disease

Mutant huntingtin protein interferes with mt import: Starves neurons of essential ATP Inhibition of mitochondrial protein import by mutant huntingtin Hiroko Yano1–4, Sergei V Baranov1, Oxana V Baranova1, Jinho Kim1, Yanchun Pan2, Svitlana Yablonska1, Diane L Carlisle1, Robert J Ferrante1, Albert H Kim2,3,5 & Robert M Friedlander1,6

Transmembrane Transport between Cytosol and Peroxisomes

Peroxisomes: Organelles for Oxidation Reactions Oxidation of very long chain and branched fatty acids Oxidation of cholesterol to bile acids (in liver) Oxidation (detoxification) of metabolic intermediates; i.e., ethanol oxidation to acetaldehyde Decomposition of hydrogen peroxide (catalase-mediated decomposition to water and oxygen) Synthesis of plasmalogens (membrane lipids found in myelin) catalase crystals

Peroxisome precursor vesicles bud from the ER

Membrane protein integrase? membrane protein receptor/transporter Peroxisomal proteins are imported from the cytosol after their synthesis is completed PTS1 receptor PTS2 receptor Membrane protein integrase? membrane protein receptor/transporter Free polyribosomes Protein translocator

Peroxisomes : IMPORTANCE Zellweger Syndrome: Rare autosomal recessive disease (1 in 50,000) caused by mutations in genes encoding peroxisome biogenesis factors; Absence of peroxisomes causes leukodystrophies (loss of white matter in CNS, due to fewer plasmalogens Absence of peroxisomes also causes accumulation of fatty acids, in macrophages, these accumulated lipids impair function, especially in CNS macrophages (this is very similar to a lysosomal storage disorder)

The correct answer is (D). Conversion of ethanol to acetaldehyde takes place in peroxisomes and smooth ER microsomes (via ADH (alcohol dehydrogenase)). Subsequent conversion of acetaldehyde to acetate takes place in mitochondria (via ALDH2 (alcohol dehydrogenase 2). Disufiram (antabuse) blocks the second reaction (to nontoxic acetate), causing accumulation of toxic acetaldehyde.

Transmembrane Transport between Cytosol and Endoplasmic Reticulum CO-TRANSLATIONAL TRANSPORT

Reconstruction of the rough and smooth endoplasmic reticulum

About half of protein synthesis is directed to the ER

Synthesis of a lumenal (often secreted) protein

Synthesis of a single-pass transmembrane protein

Synthesis of a double-pass transmembrane protein

Synthesis of a multi-pass transmembrane protein (7-transmembrane G-protein coupled receptors)

A smaller proportion are glycosylated on serine or threonine residues Proteins in the lumen of the ER are glycosylated on asparagine residues in the sequence -Asn-[X]-[Ser/Thr]- A smaller proportion are glycosylated on serine or threonine residues

Protein Folding and Misfolding in the ER: Membrane and secreted protein folding is challenging. Mis- or un-folded proteins can build up in the ER, especially in secretory cells (endocrine and some immune cells). This causes an UNFOLDED PROTEIN RESPONSE.

UNFOLDED PROTEIN RESPONSE : The sensors Indeed there are additional quality control mechanisms in place within the ER. These involve three sensors for misfolded proteins, IRE1, PERK, ATF6. Each work to maintain ER homeostasis, by signalling to the nucleus for increased expression of proteins that can improve ER client protein folding.

UNFOLDED PROTEIN RESPONSE: Homeostatic mechanism

Chronic unfolded protein response : IMPORTANCE Diabetes: Constitutive insulin secretion; UPR activation; apoptosis of islet cells; insulin dependent diabetes Several neurodegenerative diseases (Alzheimers, Parkinsons, Huntingtons, Amyotrophic Lateral Sclerosis (ALS)); UPR activation, proteinopathies. Specific examples: Alpha anti-trypsin deficiency

Protein Folding and Misfolding in the ER: Proteins failing to fold up properly are retro-translocated back into the cytosol and degraded by proteasomes. The desired proteins, therefore, never get “past” the ER IMPORTANCE: Cystic fibrosis Various lysosomal storage diseases (Gaucher disease)