1. Chapter 4. Protein sorting and vesicle trafficking

2. Fig.1-1. (A) A diagramatic representation of mesophyll leaf cell.
(B) Thin section TEM through a meristematic root tip cell.

3. The machinery of protein sorting
1.1. Protein sorting requires peptide address labels and
protein-sorting machinery

4. Table1. Peptide targeting domains for transport to different organells
Organelle Adress labels (targeting domain)
ER Signal peptide (SP)
Chloroplast Transit peptide
Mitochondrion presequence
Nucleus Nuclear localization signal (NLS)
Peroxysome Peroxysome targeting signal (PTS)
Vacuole Vacuolar sorting signal (VSS)

5. 1.2. To reach its destination, a protein often has to cross
at least one membrane

6. 1.3. Protein sorting can be a multistep process
requiring more than one targeting domain.

9. 2. Targeting proteins to the plastids
1. Transport of proteins into chloroplast involves a removable transit peptide.

11. 2.2. To enter chloroplasts, proteins pass through a proteinaceous channels
with the aid of molecular chaperones.

12. 2.3. Targeting into thylakoids required a bipartite transient peptide
and may follow three different paths from the stroma.
Toc
Toc
Toc:
translocon of the outer envelope membrane
Tic:
translocon of the inner envelope membrane
Tic
Tic

13. 3. Transport into mitochondria and peroxysomes
3.1. Transport into mitochondria resembles chloroplast import
but relies on different targeting domains, called presequences,
and a different import apparatus.

15. Translocation of mitochondria proteins: Similar in chloroplast
Tic
Tic

16. Sorting of mitochodria innner membrane protein
Cytosol  matrix
matrix  Intermembrane space
Intermembrane space  inner membrane

17. 3.2 Uptake of proteins by peroxysomes involoves removable
or intrinsic peroxysome targeting signals.
PTS: (peoxysome targeting signal)
PTS1
Consists of tripeptide (Ser-Lys-Leu, SKL) or closely related variants
Located in the C-terminus of many proteins
Not removed after translocation
PTS2
Consists of a cleavable N-terminal targeting domain
Is used by a subset of peroxysomal matrix proteins
Passenger proteins fused to PTS-like seq. are directed into peroxysomes

18. The peroxysome targeting signal Ser-Lys-Leu (SKL) directs
a bacterial enzyme to peroxysomes.
Chloramphenicol acethyltransferase (CAT) from bacteria
Peroxysomal enzyme, a catalase
CAT with the signal SKL to the C terminus

19. 4. Transport in and out of the nucleus
4.1.
The nuclear pore is the site for macromolecular movement into and out of nucleus.

20. The surfaces of the nuclear envelope
(obtained by freeze-fraction and freeze etching of a specimen)

21. Nuclear pore complexes contain glycoproteins
Wheat germ agglutinin: a lectin that blocks protein import into vertebrate nuclei.

22. 4.2. Nuclear localization signals target proteins to the nucleus.
NLS generally
1) contain several residues of Arg and Lys
2) and may also have residues such as proline that disrupt helical domains.
Virtually all classical NLSs may be categorized into one of three classes.
Viral NLS: (PKKKRKV)
MSERKRREKL : SV40-like
2) Bipartite NLS: contain basic residues and are separated by a spacer (10 or more)
RKRKESNRESARRSRYRK
3) The basic residues are separated by three hydrophobic residues.
MISEAIRKAIGKR : MATa2-like

23. (C) Nuclear envelope binding (D) nuclear import
4.3 Nuclear import can be studied both in vivo and in vitro.
(A) GUS staining (B) DAPI staining
(C) Nuclear envelope binding (D) nuclear import
Confocal microscopy

24. 4.4 Nuclear import is controlled by several mechanisms,
providing an additional level of regulation