BIOC 212 Lecture Notes - Lecture 17: Microtubule Organizing Center, Cytosol, Autophagosome
29 May 2018
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6- Intracellular Trafficking
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Endosome Maturation
• Attachment of single ubiquitin onto a lysine is major signal for receptor endocytosis
o Receptor on PM --> endosome
• Some TM proteins are marked for endocytosis by modification with mono-ubiquitin
at PM
o Not poly-ubiquitin, no proteasomal degradation
§ Attached to lysine in the same way
§ Different type of E3 ligases mediate attachment
o Recognition by CCV adaptors
§ Recognized by adaptors in the formation of clathrin coated vesicles
§ Gives targeting to endosome
o If remove Ub, get recycled back to PM
o If ubiquitin is not removed, proteins are not recycled to PM
§ Stay there and move forward to mature endosome & multi-vesicular
body
• Using mono-Ub for two targeting steps
o Endocytosis at PM
o At the endosome, for whether should recycle or go forward
• Early endosomes mature into MVBs (multivesicular bodies) by invaginating and
pinching-off membrane
o Committed step; once in a multi-vesicular body, cannot go back to the PM or
Golgi
§ Not reversible
o Must go forward to the lysosome
o MVB contents cannot be recycled to PM anymore
• MVBs fuse with lysosomes and other vesicles to give final compartment
MVB Formation
• MVB invagination: a series of ESCRT protein complexes shape and pinch off
vesicles into the lumen of an endosome
o Same main four steps as for vesicle formation
o Membrane protrudes into lumen, forming a bud, and then membranes on
either side are pulled together to form entirely enclosed vesicle
• ESCRT-0 binds PI(3)P and collects mono-Ub cargo proteins, provides binding site
for ESCRT-I
o Initiation & adaptor step
• ESCRT-I and II form the neck of the bud, ESCRT-II provides binding site for
ESCRT-III
o Physically shaping the membrane
3. ESCRT-III forms oligomers to pinch off the bud to form vesicle
o Form vesicle that is entirely enclosed by the lumen
o Pinching it off
ESCRT-0 & Ub Binding
• Main interactions are where PI phosphates & mono-Ub
o Also have interaction with clathrin
o Clathrin does not form an actual cage; forms a partial lattice on the surface
o Helps ESCRT-0 form clusters in which gather up mono-Ub cargo in local area
in which form a bud
• Clathrin binds multiple ESCRT-0 complexes to form clusters (microdomains) but
not complete vesicles
o Microdomain since is a type of lateral organization in membrane
• Ub-binding domains (UIM, UEV, GLUE) in ESCRT-0 to II subunits
o All bind mono-Ub
o Passing off the ubiquitinated cargo from 0 to I to II
• PI(3)P-binding domains (FYVE, GLUE) in ESCRT-0 and II subunits
o Always maintain interaction with membrane
ESCRT-I and II
• ESCRT-I forms a long structure which could start to deform membrane
o Long and extended structure
o Form long bars across the membrane that deforms the membrane by pushing
it inwards a bit
o Reach across neck of bud, or directly bend the membrane
• ESCRT-II binds at the intersection between multiple ESCRT-I
o ESCRT-II is more compact and anchors ESCRT-III
o ESCRT-III is what gives the pinching off
ESCRT-III
• Contains homologous subunits (Snf7, Vps2, Vps24)
• Homodimers of Snf7, and heterodimers of Vps2/Vps24, assemble into a long
filament
o When inactive, exist as dimers in the cytosol
o When initiate formation, have ESCRT-II as a platform; start forming a long
filament composed of multiple dimers
o Filament is what gives pinching off
• Additional proteins are a DUB (Doa4/UBPY) and adaptors to AAA disassembly
factor (Vps4) are associated with ESCRT-III
Document Summary
Endosome maturation: attachment of single ubiquitin onto a lysine is major signal for receptor endocytosis, receptor on pm --> endosome, some tm proteins are marked for endocytosis by modification with mono-ubiquitin at pm, not poly-ubiquitin, no proteasomal degradation. Attached to lysine in the same way. Different type of e3 ligases mediate attachment: recognition by ccv adaptors. Recognized by adaptors in the formation of clathrin coated vesicles. If remove ub, get recycled back to pm. If ubiquitin is not removed, proteins are not recycled to pm. Not reversible: must go forward to the lysosome, mvb contents cannot be recycled to pm anymore, mvbs fuse with lysosomes and other vesicles to give final compartment. Initiation & adaptor step: escrt-i and ii form the neck of the bud, escrt-ii provides binding site for. Escrt-iii: physically shaping the membrane, escrt-iii forms oligomers to pinch off the bud to form vesicle, form vesicle that is entirely enclosed by the lumen, pinching it off.
