endocytosis 3

exit from Golgi

• trans Golgi network as sorting station for outbound cargo and interface for Golgi and endocytic pathway

• direction of protein exit is anterograde

• secretion to plasma membrane or cargo transport to endosome

• transport routes can be direct or indirect

• transport of cargo via carriers (vesicles, tubular intermediates)

constitutive secretion

• occurs in all cells

• supplies plasma membrane with newly synthesized lipids and proteins

• immediate fusion with plasma membrane without signal

regulated secretion

• only in some cells

• cargo: hormones, neurotransmitters, digestive enzymes

• fusion with plasma membrane upon signal

dense core vesicles

• found in neurons and endocrine cells

• contain large neuropeptides rather than low molecular neurotransmitters

packaging of cargo for regulated secretory pathway

• cargo aggregation at trans Golgi network

• vesicle containing cargo buds from TGN

• immature secretory vesicle undergoes membrane and lumenal content recycling

• secretory vesicle acidified

• mature secretory vesicle waits for signal at plasma membrane

  • signal causes Ca²⁺ influx

secretion in polarized cell

• apical versus basolateral targeting

• tight junctions prevent lateral migration of transmembrane proteins, prevent mixture of apical and basolateral constituents

• proteins are sorted based on signals that are recognized by receptors/carriers at TGN

• targeting routes can be direct or indirect

secretion to apical membrane

• signals are pleiomorphic

  • O-linked glycosylation

  • N-linked glycosylation

    • weak signal which can be overridden by basolateral targeting signals

  • protein motifs

  • GPI anchor

• GPI anchors normally associate with lipid rafts at TGN

tubular transport intermediates

• Golgi to apical cell surface in polarized epithelial cells

• tubules use kinesins to move

• mechanism of cargo selectivity probably involves partition of cargo into lipid rafts

secretion to basolateral membrane

• signals are less complex than apical sorting signals

• tyrosine-based (NPxY or YxxO) or dileucine (D/ExxLL) motifs embedded into cytoplasmic tail of proteins

• sorting of most proteins requires clathrin

• adaptor protein is AP1B complex

• same cytoplasmic signals can be used to re-target cargo back to the basolateral membrane after its endocytosis

general structure of APs

• made of four domains, specific to type and tissue

• can bind receptors via tyrosine and dileucine motifs

• binds clathrin through ear

AP-1

• involved in trafficking to cell surface (AP-1b) and endosomes (AP-1a)

• subtypes differ only in μ subunit

AP-3

involved in trafficking to endosomes

AP-4

on Golgi

transport from trans Golgi network to endosomes

• direct pathway: endosome → lysosome

• indirect: plasma membrane → endocytosis → lysosome

• catabolic proteins: activators, soluble and membrane hydrolases

• protective: membrane proteins are N-glycosylated to protect cell interior

• multiple transporters and channels in lysosomal membrane

lysosomal membrane proteins

• LAMPs and LIMPs

• transport relies on dileucine-based motifs (DXXLL or [DE]XXXL[LI]) and tyrosine-based motifs (YXXO)

• motifs interact with GGA or AP-3 complex

lysosomal acid hydrolases

• ~40 types

• function in low pH (acidic)

• most need to be activated by proteolytic cleavage

structure and function of GGAs

• made of 4 domains

• able to bind receptor tail motif via VHS domain

• binds ARF via GAT domain

• binds clathrin via hinge and terminal ear domain

mannose-6-phosphate tag

1. at cis Golgi, GlcNAc-phosphotransferase transfers UDP-GlcNAc to N-linked oligosaccharide attached to lysosomal hydrolase

   • mannose residue is in N-linked oligsaccharide

2. at medial Golgi, phosphodiester alpha-GlcNAcase removes GluNAc to leave M6P

ICD disease

• mutation of GlcNac phosphotransferase

• M6P recognition signal not exposed

• hydrolase not recognized by M6PR

types of lysosomal storage disorder

• defects in glycan degradation

• defects in lipid degradation

• defects in protein degradation

• defects in lysosomal transporters

• defects in lysosomal trafficking

lysosomal storage disease

• characterized by enlarged lysosomes with excessive material

• nervous system is particularly vulnerable to damage

• individuals normally born healthy, with progressive development of symptoms

• disease are inherited in an autosomal-recessive fashion, except for X-linked recessive

• speed and severity of symptoms depends on:

  • which cells affected

  • genetic background of individual

  • environmental factors

  • type of waste product accumulated

inclusion-cell disease (I-cell disease)

• most severe form of lysosome storage disease

• rare, inherited, recessive

• defect in transferring UDP-GlcNAc to hydrolases by N-GlcNAc phosphotransferase (defects in hydrolases trafficking)

• most hydrolases absent from lysosomes

• caused by frameshift or premature stop codon in UDP-N-acetylglucosamine N-acetylglucosaminyl-1-phosphotransferase

• major symptoms: severe delayed development, short stature, coarse facial features, dysostosis multiplex, cardiomyopathy

• clinical diagnosis: high lysosomal enzyme activity in serum, molecular genetic screening

general symptoms of I cell disease

• characterized by accumulation of excessive amounts of acid mucopolysaccharides in fibroblast, connective tissue, connective tissue cells

• abnormal accumulation of undigested sphingolipids and/or glycolipids in the lysosomes of visceral fibroblasts, macrophages, nerve cells

• results in mental retardation and skeletal changes, most notably dysostosis multiplex

dysostosis multiplex

• mucopolysaccharides due to deficiency of alpha-I-iduronidase

• accumulation of abnormal intracellular material and excretion of dermatan sulfate and heparan sulfate in urine

• severe abnormality in development of skeletal cartilage and bone

• dwarfism

• deformed limbs

• limitation of joint motion

• spade-like hand

• corneal clouding

• hepatosplenomegaly

• mental retardation

• gargoyle-like face

M6P receptor

• two types (CI and CD)

• transport most lysosomal hydrolases

• some overlap in cargo recognition between two types

• cytoplasmic tails contain sorting signals (dileucine and tyrosine)

• dileucine signal recognized by GGA

adaptor GGA

• recognizes M6PR

• contains 4 domains

  • VHS recognizes DXXLL signals, binds to both forms of M6PR and sortilin

  • GAT binds to GTP-bound Arf-1

  • hinge binds to clathrin

  • GAE binds accessory proteins

Gaucher’s disease

• beta-glucocerebrosidase / GBA

• major symptoms: hepatosplenomegaly, anemia, skeletal disease, dementia, convulsions

• clinical diagnosis: bone marrow aspirate, beta-glucocerebrosidase activity

Gaucher cell

• contains abundant granular or fibrillary, blue-gray cytoplasm with wrinkled tissue paper-like appearance

• saposins B and C assist various lysosomal enzymes in catabolism of sphingolipids

sortilin

• receptor that uses GGA adaptor proteins

• involved in transport of activator protein prosaposin as well as a number of other activator proteins and hydrolases

• does not require M6P tag for cargo recognition

• directly binds to cargo proteins

• at C-terminus, motifs recognized by GGA and GGA-AP-1 complex

• binds to C-terminus of prosaposin

• saposin A-D for sphingolipid hydrolysis, wither as solubilizers or liftases

truncated GGA

lacks hinge and ear domains to interact with clathrin

targeting of membrane proteins to lysosomes

• LEP, LAMPs, LIMPs

• occurs normally even if GGA not functioning

• depends on clathrin

AP-3 mutant cells

membrane proteins remain on plasma membrane instead of lysosomes

transport of most soluble hydrolases

• generally done by M6PR

• M6PRc uses GGA adaptor proteins to enter clathrin coated cargo vesicles

transport of lysosomal membrane proteins

• LEP 100, LIMP 1-4, LAMPs are membrane glycoproteins

• go to lysosomes from Golgi apparatus using adaptor proteins

• transport of lysosomal associated protein (LAMP) is mediated by AP-3

retromer

• composed of 2 multisubunit complexes

  • heterodimer sorting nexins (Snx1/2) → induce and sense membrane curvature

  • heterotrimeric Vps35-29-26 (cargo-selective complex)

• recruitment based on two conditions

  • bind to PI3P

  • bind to Rab7

• believed to be clathrin independent

• involved in return of M6PR and sortilin from late endosome to Golgi

• produces tubular buds with unusual morphology